Selective preventives/remedies for progressive lesions after organ damage

ABSTRACT

Drugs for preventing and/or treating progressive lesions after organ damage without inhibiting the function of the organ or the regeneration function thereof, by selectively regulating the induction of cytotoxic effecter macrophages which are induced into damaged organ tissues in response to chemokines or cytokines expressed depending on the type of the damaged organ tissues.

This application is a 371 of PCT/JP01/02513 filed Mar. 27, 2001.

TECHNICAL FIELD

Organs in organism may be damaged, for example, by blood flow disorder,ischemia reperfusion injury, hypertension, hyperglycemia, hyperlipemia,pharmaceutical agent or viral infection. Damaged organ tissues show anyof responses of necrosis, natural death or self-regeneration dependingupon the degree of the damage. Immune system deeply participates in sucha response and some of macrophages which are carriers of such immuneparticipate in self-regeneration of organ tissues while othersparticipate in necrosis or natural death of organ tissues. The latter,i.e. that which shows cytotoxicity in a functional manner among the muchdifferentiated macrophages, is called effector macrophage and such aneffector macrophage further worsens the damage after the above-mentioneddamage and causes a progressive lesion after the organic damage.

The present invention relates to a pharmaceutical in which induction ofeffector macrophage which is a cause of progressive lesion noted aftersuch an organic damage is selectively suppressed whereby the saidprogressive lesion is prevented and/or treated without inhibiting thefunction and regeneration process of the organ, and also relates to atherapeutic method using the same. The present invention further relatesto a screening method for compounds which can be the saidpharmaceutical. The present invention still further relates to novelγ-lactone derivatives useful as the said pharmaceutical.

With regard to one of the progressive lesions noted after theabove-mentioned organic damages, fibrosis of tissue cells may beexemplified. Such a fibrosis of tissue cells is caused by effectormacrophage showing cytotoxic property the same as above. The novelγ-lactone derivatives according to the present invention are able toselectively suppress the induction of the said effector macrophage. As aresult of utilization of such an action, the present invention alsorelates to a pharmaceutical as a fibrosis inhibitor containing the saidnovel γ-lactone derivative.

Rejection, particularly chronic rejection, upon transplantation ofallogenic or xenogenic organ cells is caused by the same mechanism inthe progressive lesion after organic damage or, at least similar,mechanism to that. The novel γ-lactone derivatives according to thepresent invention selectively suppress the induction of effectormacrophage causing the induction of allograft rejection, particularlychronic rejection, upon allogenic or xenogenic cell, tissue or organtransplantation whereby they show an immunosuppressive action only inthe organ tissues showing the rejection. As a result of utilization ofsuch an action, the present invention also relates to a pharmaceuticalas an immunosuppressant containing the said novel γ-lactone derivative.

BACKGROUND OF THE INVENTION

When an organ in organisms is damaged, for example, by blood flowdisorder, ischemia reperfusion injury, hypertension, hyperglycemia,hyperlipemia, pharmaceutical agent or viral infection, there works anorganism defense mechanism by, for example, T lymphocytes, macrophages,NK cells, fibroblasts, B lymphocytes and antibodies, complements, etc.Particularly, T cells and macrophages have an important participation inthe said organism defense mechanism.

Damaged organ tissues show any of the responses of necrosis, naturaldeath or self-regeneration depending upon the degree of the damage. Atthat time, chemokines, cytokines, etc. are expressed from the saidtissues to promote self-proliferation and regeneration and, at the sametime, organism defense mechanism of the host is activated as well. Ithas been presumed that the outcome whether the damaged tissues result innecrosis or natural death by way of atrophy and fibrosis or they resultin regeneration of tissues is the result of the interaction of both.

For example, there are several reports for the studies that, in thedamaged renal tissue lesions, the damaged renal tissue cells expresstissue-specific chemokine, cytokine or adhesion factor depending uponthe damaged area, etc. whereby the response is exhibited.

Wada, et al. reported that, in human crescentic glomerulonephritis,MIP-1α of chemokine is expressed in glomerular cellular crescents in anacute stage while, in interstitial tissues of the cases of fibrouscrescents in a chronic stage, MCP-1 is expressed (Wada T., Furuichi K.,Segawa-Takeda C., Shimizu M., Sakai N., Takeda S. I., Takasawa K., KidaH., Kobayashi K. I., Mukaida N., Ohmoto Y., Matsushima K., Yokoyama H.:MIP-1α and MCP-1 contribute to crescents and interstitial lesions inhuman crescentic glomerulonephritis. Kidney Int., 56: 995-1003, 1999.

Matsuda, et al. reported that, in model rats suffering from crescenticglomerulonephritis, P-secretin and L-secretin are expressed in adiscriminated manner in glomerular endothelial cells and in interstitialtissues from urinary tubule, respectively (Michihiro Matsuda, KenichiShikata, Daisuke Ogawa, Shinichi Okada, Yasushi Shikata, Atsushi Wada.and Hiroshi Makino: Mechanism of Induction of Infiltration of Leucocytesto Renal Tissues by Secretin, Nippon Jinzo Gakkaishi, 42: 213, 2000).

Tesch, et al. reported that, when nephrotoxic serum nephritis model wasprepared in MCP-1 knockout mice and expression of MCP-1 was checked, theexpression of MCP-1 in the damaged site of urinary tubule was weak ascompared with a wild type while the expression of MCP-1 in theglomerular lesion had no difference from the wild type. They alsoreported that, in the knockout mice where expression of MCP-1 was weak,infiltration of macrophage decreased whereby MCP-1 showed the resultthat it participated in migration of macrophage while, in terms ofdegree of proteinuria in an acute stage, there is no difference betweenboth and there was no relation between tissue damage and macrophageinfiltration in an acute stage (Tesch G. H., Schwarting A., KinoshitaK., Rolins B. J., Kelly V. R.: Monocyte chemoattractant protein-1promotes macrophage-mediated tubular injury, but not glomerular injury,in nephritic serum nephritis, J. Clin. Invest. 1999, 103: 73-80.).

Not only in kidney but also in exocrine gland and islet of Langerhans ofpancreas, there is noted a difference in chemokine-productive responseconcerning the lesion after the organ tissue damage corresponding to thedamaged tissue.

Cameron, et al. reported that, in the mice where nonobese type Idiabetes was spontaneously occurred, promotion of the expression ofMIP-1α and lowering of the expression of MCP-1 were noted amongchemokines in islets of Langerhans and that there was a correlationbetween damage of islets of Langerhans and onset of diabetes (Cameron M.J., Arreaza G. A., Grattan M., Meagher C., Sharif S., Burdick M. D.,Strieter R. M., Cook D. N., Delovitch T. L.: Differential expression ofCC chemokines and the CCR5 receptor in the pancreas is associated withprogression to type I diabetes, J. Immunol., 2000, 165: 1102-10).

In the meanwhile, Andoh, et al. investigated the expression ofchemokines in pancreatitis tissues of human acute pancreatitis cases.They reported that, in acute pancreatitis, expression of MIP-1α was notnoted but expression of MCP-1, IL-8 and RANTES was noted in exocrine,acinar and ductal interstitial pancreas tissues (Andoh A., Takaya H.,Saotome T., Shimada M., Hata K., Araki Y., Nakamura F., Shintani Y.,Fujiyama Y., Bamba T.: Cytokine regulation of chemokine (IL-8, MCP-1,and RANTES) gene expression in human pancreatic periacinarmyofibroblasts, Gastroenterology, 2000, 119: 211-9).

As mentioned above, it was suggested in human clinical studies andexperimental study models using animals that, in progressive lesionnoted after the organic damage, effector macrophage participated thereincorresponding to the damaged tissues.

Up to now, therapy of steroids, etc. has been carried out forprogressive lesion after organic damage. However, since steroidalpreparations non-selectively suppress the macrophage, they also suppressthe response of even the macrophage participating in the reaction fortissue regeneration at the same time whereby organism defensivemechanism including the regeneration is lessened. In addition, newtissue damage is induced resulting in lesion and, as a result, there isa problem that effector macrophage mediated by the expression ofchemokines and cytokines is actively induced whereby the inherent lesionis further worsened. As such, the conventional therapy of steroids hasno selectivity in the action, and administration of high dose isnecessary for the therapy of the lesion whereby the side effect isremarkable. In addition, there is a difficulty that a continuous therapyby steroids for a long period is accompanied by a severe side effect.

On the other hand, like the progressive lesion noted after theabove-mentioned organic damage, organism defense mechanism by T cells,macrophages, etc. participates in rejection in the transplantation oforgan, skin or the like. Until now, there have been known many compoundshaving an immunosuppressive action and, for example, compoundsrepresented by the formula (8)

(in which R¹ is an optionally substituted phenyl group; R² is anoptionally esterified carboxyl group; and X is oxygen atom or optionallyoxidized sulfur atom) have been known to be useful as a γ-lactoneimmunosuppressant (Japanese Patent Laid-Open No. 04/338,331). However,it is to be still improved so as to show a selective action to targetorgans or tissues or so as to show stronger immunosuppressive action.

DISCLOSURE OF THE INVENTION

Effector macrophage expresses chemokine receptors or β2 integrinreceptors, etc. corresponding to the lesion inherent to the tissuesafter the organic damage via either T-lymphocyte-independent orT-lymphocyte-dependent response and is selectively induced to andactivated in lesion being mediated by them whereby said effectormacrophage causes a progressive lesion after the tissue damage. Anobject of the present invention is to provide a pharmaceutical in whichexpression and function of chemokine receptors such as CCR2, CCR3 orCCR8, β2 integrin receptors such as CD11b/CD18 or other receptors aresuppressed to selectively suppress the induction of the above-mentionedeffector macrophages whereby progressive lesion after organic damage isprevented and/or treated without inhibiting the function of the organand the regeneration function and also to provide a therapeutic methodusing the same. Since the pharmaceutical according to the presentinvention is able to suppress the induction of effector macrophagescausing the progressive lesion after organic damage in a morelesion-selectively manner, it is possible to use in a high dose and, dueto the selectivity of the said pharmaceutical, undesirable side effectcan be avoided even when it is used in a high dose. Therapy for a longperiod using the said pharmaceutical is now possible as well. Further,when the lesions are different resulting in progressive lesion after theorganic damage in plural tissues, it is possible to carry out moreselective and more effective therapy by combination use of the abovepharmaceuticals showing different selectivity. That is an advantagewhich is not noted in the conventional steroidal agents and knownγ-lactone immunosuppressants having no selectivity. The presentinvention also aims to provide useful and novel γ-lactone derivatives.

Another object of the present invention is to provide a method forscreening the compounds which do not substantially suppress themacrophage participating in tissue regeneration and cause theprogressive lesion after the above-mentioned organic damage.

Still another object of the present invention is to provide a method forinducing the effector macrophage which causes the progressive lesionafter the above-mentioned organic damage.

Further object of the present invention is to provide a pharmaceuticaluseful as a fibrosis inhibitor preventing from the fibrosis which is oneof lesions inherent to the tissues after the organic damage or, to bemore specific, to provide a fibrosis inhibitor containing theabove-mentioned novel γ-lactone derivative.

Still further object of the present invention is to provide apharmaceutical which are useful as an immunosuppressant or, to be morespecific, to provide an immunosuppressant which is an immunosuppressantcontaining the above-mentioned novel γ-lactone derivative and shows aselective immunosuppressive action to target organs or tissues unlikethe known γ-lactone immunosuppressants.

As mentioned above, damaged tissue cells express chemokine, cytokine oradhesion molecule inherent to the tissue. Depending upon type or degree(amount) of the expression or upon defense mechanism of organism, theexpressed chemokine, cytokine, etc. either promote self-proliferation orregeneration of the damaged tissues or subject the damaged tissue tonecrosis, natural death or degeneration. Here, effector macrophageshowing cytotoxic property is selectively induced corresponding to typeor expressed amount of the said chemokine and cytokine so that thedamaged tissues are subjected to necrosis, natural death ordegeneration. To be more specific, effector macrophage is selectivelyinduced to the damaged organ tissues (lesion tissues) by chemokinereceptors corresponding to the said chemokine expressed in the damagedorgan tissues (lesion tissue) or by β2 integrin, etc. corresponding to aligand expressed on the said organ tissues (lesion tissues). The inducedeffector macrophage recognizes the damaged tissues, acts on the saidtissues in a cytotoxic manner and induces the progressive lesion afterthe organic damage. On the other hand, the effector macrophage does notrecognize the normal tissue wherein chemokine and cytokine whichstimulate for inducing and activating the said effector macrophage arenot expressed and, as a result, the normal tissue is not damaged.

Accordingly, if induction of effector macrophage in the damaged organtissues can be selectively suppressed by way of suppression ofexpression and function of chemokine receptor such as CCR2, CCR3 andCCR8, β2 integrin receptors such as CD11b/CD18 and other receptors,action of the effector macrophage in a cytotoxic manner to the damagedorgan tissues can be prevented and, in addition, induction of themacrophage participating in the tissue regeneration is not suppressed sothat lowering of the defense ability of the organism is not notedwhereby it has been found that progressive lesion after organic damagecan be prevented, mitigated or treated.

When a compound which is able to selectively suppress the induction ofeffector macrophage as such is used as a pharmaceutical, there isreduced the side effect such as that defense of organism isunnecessarily lowered or new tissue damage is induced. In addition, itis possible to result in recovery and regeneration of the tissue withoutan unnecessary lowering of defense of organism and, therefore, it is nowpossible to continuously carry out the therapy for a long period.

Ishibashi who is one of the inventors of the present invention hascarried out a further investigation for the progressive lesion in kidneyon the basis of the above finding and has obtained an unexpected findingthat a compound which suppresses the induction of effector macrophagecaused by contact of human peripheral blood mononuclear cellsabbreviated as PBMC with lipopolysaccharide suppresses the progress ofglomerular lesion of kidney and that a compound which suppresses theinduction of effector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC suppresses the progress of progressivelesion of tubulointerstitial tissue after renal damage.

Ishibashi who is one of the inventors of the present invention has alsocarried out an investigation for progressive lesion in pancreas andobtained an unexpected finding that a compound which suppresses theinduction of effector macrophage caused by contact of human PBMC withlipopolysaccharide suppresses the progress of lesion in islets ofLangerhans of pancreas and that a compound which suppresses theinduction of effector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC suppresses the progress of lesion ofexocrine, acinar and ductal interstitial tissues of pancreas.

Here, when lesion of islets of Langerhans of pancreas progresses,diabetes mellitus is able to occur. When diabetes mellitus occurs,diabetic nephropathy which is a glomerular lesion is able to occur as acomplication thereof. Ishibashi who is one of the inventors of thepresent invention has further obtained an unexpected finding that acompound which suppresses the induction of effector macrophage caused bycontact of human PBMC with lipopolysaccharide is also able to preventand/or treat the onset of diabetic glomerular lesion (another name:diabetic nephropathy) together with the onset of diabetes mellitus.

Ishibashi who is one of the inventors of the present invention hasfurthermore obtained an unexpected finding that a compound whichsuppresses the induction of effector macrophage caused by contact ofhuman PBMC with mitomycin-treated human PBMC is also able to preventand/or treat the onset of lesion of tubulointerstitial tissues which isa complication of pancreatitis together with onset of pancreatitis whichis lesion of exocrine, acinar and ductal interstitial tissues ofpancreas.

The present inventors have carried out an investigation for thecompounds which are able to selectively suppress the induction ofeffector macrophage which is caused corresponding to lesion inherent totissues after the organic damage and, as a result, they have found thatnovel γ-lactone derivatives represented by the following formulae offrom (1) to (7) have such an action.

The present inventors have further found that, since fibrosis of tissuesis one of the progressive lesions after the organic damage, the novelγ-lactone derivatives represented by the following formulae of from (1)to (7) are also useful as inhibitors for fibrosis.

With regard to the rejection at the transplantation of organ cells ofallogeneic or xenogenic type, there are acute rejection and chronicrejection and it has been known that, particularly in the chronicrejection, not only immunological factors but also non-immunologicalfactors such as damage by pharmaceuticals, ischemia reperfusion injury,viral infection, blood flow disorder and exclusion of cells can be acause. On the other hand, the above-mentioned progressive lesion afterorganic damage also results from the organic damage, which is a trigger,such as damage by pharmaceuticals, ischemia reperfusion injury, viralinfection, blood flow disorder and exclusion of cells. Accordingly,chronic rejection is caused by a mechanism which is the same as or atleast similar to the progressive lesion after the organic damage.Therefore, it has been found that novel γ-lactone derivativesrepresented by the following formulae of from (1) to (7) which are ableto selectively suppress the induction of effector macrophage inducedbeing correspondent to the lesion inherent to the tissues after organicdamage are useful as an immunosuppressant for the prevention or thetherapy of rejection upon of allogeneic or xenogenic cell, tissue ororgan transplantation, particularly as an immunosuppressant to chronicrejection.

Thus, the present invention relates to the followings.

(1) A pharmaceutical composition, which comprises a compound suppressingthe induction of effector macrophages.

(2) A preventive and therapeutic pharmaceutical selectively toprogressive lesion of organic damages which comprises a compoundsuppressing the induction of effector macrophages.

(3) A pharmaceutical for prevention and/or therapy of glomerular lesionof kidney, which comprises a compound suppressing the induction ofeffector macrophages caused by contact of human PBMC withlipopolysaccharide.

(4) A pharmaceutical for prevention and/or therapy of progressivetubulointerstitial lesion after renal damage, which comprises a compoundsuppressing the induction of effector macrophages caused by contact ofhuman PBMC with mitomycin-treated human PBMC.

(5) A pharmaceutical for prevention and/or therapy of lesion of isletsof Langerhans of pancreas, which comprises a compound suppressing theinduction of effector macrophages caused by contact of human PBMC withlipopolysaccharide.

(6) A pharmaceutical for prevention and/or therapy of lesion ofexocrine, acinar and ductal interstitial tissues of pancreas, whichcomprises a compound suppressing the induction of effector macrophagescaused by contact of human PBMC with mitomycin-treated human PBMC.

(7) A pharmaceutical for prevention and/or therapy of diabetes mellitusand diabetic glomerular lesion, which comprises a compound suppressingthe induction of effector macrophages caused by contact of human PBMCwith lipopolysaccharide.

(8) A pharmaceutical for prevention and/or therapy of pancreatitis andlesion of interstitial tissues of urinary tubule which is a complicationof pancreatitis, which comprises a compound suppressing the induction ofeffector macrophages caused by contact of human PBMC withmitomycin-treated human PBMC.

(9) A method for prevention and/or therapy of glomerular lesion ofkidney, lesion of islets of Langerhans of pancreas or diabetes mellitusand diabetic glomerular lesion, which comprises using a pharmaceuticalcontaining a compound suppressing the induction of effector macrophagescaused by contact of human PBMC with lipopolysaccharide.

(10) A method for prevention and/or therapy of progressivetubulointerstitial lesion after renal damage, lesion of exocrine, acinarand ductal interstitial tissues of pancreas or pancreatitis, and lesionof interstitial tissues of urinary tubule, which comprises using apharmaceutical containing a compound suppressing the induction ofeffector macrophages caused by contact of human PBMC withmitomycin-treated human PBMC.

(11) A method for screening a compound which is able to prevent,mitigate or treat glomerular lesion of kidney, lesion of islet ofLangerhans of pancreas or diabetes mellitus and diabetic glomerularlesion, which comprises measuring a suppressive action of a compound tobe tested against the induction of effector macrophage caused by contactof human PBMC with lipopolysaccharide.

(12) The method for screening according to the above-mentioned (11),wherein effector macrophages are induced by incubation of humanuntreated PBMC in RPMI 1640 medium in the presence of a compound to betested, lipopolysaccharide and human AB type serum, the said inducedeffector macrophages are brought into contact with monolayeredautologous erythrocytes and a compound showing less production of SPFC,Spontaneous Plaque-Forming Cell, as compared with the absence of thesaid compound to be tested is screened.

(13) A method for screening a compound which is able to prevent,mitigate or treat progressive tubulointerstitial lesion after renaldamage, lesion of exocrine acinar and ductal interstitial tissues ofpancreas or pancreatitis and lesion of interstitial tissues of urinarytubule, which comprises measuring a suppressive action of a compound tobe tested against the induction of effector macrophages caused bycontact of human PBMC with mitomycin-treated human PBMC.

(14) The method for screening according to the above-mentioned (13),wherein effector macrophages are induced by a mixed incubation ofmitomycin-treated human PBMC and human untreated PBMC in RPMI 1640medium in the presence of a compound to be tested and human AB typeserum, the said induced effector macrophages are brought into contactwith monolayered autologous erythrocytes and a compound showing lessproduction of SPFC as compared with the absence of the said compound tobe tested is screened.

(15) A kit for screening a compound which is able to prevent, mitigateor treat glomerular lesion of kidney, lesion of islet of Langerhans ofpancreas or diabetes mellitus and diabetic glomerular lesion, whichcomprises (a) human PBMC, (b) lipopolysaccharide, (c) human AB typeserum, (d) RPMI 1640 medium and (e) a plate to which monolayeredautologous erythrocytes are adhered.

(16) A kit for screening a compound which is able to prevent, mitigateor treat progressive tubulointerstitial lesion after renal damage,lesion of exocrine, acinar and ductal interstitial tissues of pancreasor pancreatitis and lesion of interstitial tissues of urinary tubule,which comprises (a) human PBMC, (b) mitomycin-treated human PBMC, (c)human AB type serum, (d) RPMI 1640 medium and (e) a plate to whichmonolayered autologous erythrocytes are adhered.

(17) A method for the induction of effector macrophages which are acause of glomerular lesion of kidney, lesion of islets of Langerhans ofpancreas or diabetes mellitus and diabetic glomerular lesion, whichcomprises bringing lipopolysaccharide into contact with human PBMC.

(18) A method for the induction of effector macrophages which are acause of progressive tubulointerstitial lesion after renal damage,lesion of exocrine, acinar and ductal interstitial tissues of pancreasor pancreatitis and lesion of interstitial tissues of urinary tubule,which comprises bringing human PBMC into contact with mitomycin-treatedhuman PBMC.

(19) The pharmaceutical according to any of the above-mentioned (1) to(8), which comprises a compound represented by the formula (1)

or a compound represented by the formula (2).

(20) An optical isomer γ-lactone represented by the formula (3)

or by the formula (4)

(in the formula, R²¹ is an optionally substituted naphthyl group and R²²is an optionally substituted straight or branched hydrocarbon grouphaving 1 to 6 carbon atoms) or a mixture of the above optical isomers.

(21) The optical isomer γ-lactone or a mixture of the optical isomersaccording to the above-mentioned (20), wherein R²¹ is naphthyl and R²²is methyl.

(22) A compound represented by the formula (5) or a pharmacologicallyacceptable salt thereof.

(in the formula, (a) R¹ and R² may be the same or different and each ishydrogen, an open-chain aliphatic hydrocarbon group which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; X² is O, S orNR³ in which R³ is hydrogen, oxygen, an open-chain aliphatic hydrocarbongroup which may be substituted or interrupted by an intervening group,an optionally substituted cyclic aliphatic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and n is an integer from 1 to 5 or

(b) X² is O, S or NR³; R¹, R² and R³ each is a substituent representedby the formula R¹⁰—Z—R¹¹— (in which R¹⁰ and R¹¹ may be the same ordifferent and each is an optionally substituted open-chain or cyclichydrocarbon group, an optionally substituted aryl group, an optionallysubstituted heterocyclic group or an optionally substituted condensedheterocyclic group; and Z is an intervening group); and n is an integerfrom 1 to 5).

(23) A compound represented by the formula (6) or a pharmacologicallyacceptable salt thereof.

(in the formula, R¹, X² and n have the same meaning as defined in theabove-mentioned (4); X¹ is halogen, cyano group, an optionallysubstituted mercapto group, an optionally substituted sulfo group, anoptionally substituted sulfonyl group, an optionally substitutedhydroxyl group, an optionally substituted amino group or an optionallysubstituted phosphoryl group).

(24) A compound represented by the formula (7) or a pharmacologicallyacceptable salt thereof.

(in the formula, R¹ and X² have the same meaning as defined in theabove-mentioned (4); X¹ has the same meaning as defined in theabove-mentioned (5); R⁵ and R⁶ may be the same or different and each is(a) hydrogen, a straight or branched aliphatic hydrocarbon group whichmay be substituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, or an optionallysubstituted heterocyclic group, an optionally substituted condensedheterocyclic group,

(b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in the formula,R¹⁰ and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group.), or

(c) R⁵ and R⁶ together with the carbon atom to which they are attachedform an optionally substituted aromatic ring).

(25) The pharmaceutical according to any of the above-mentioned (1) to(8), which comprises a compound mentioned in any of the above-mentioned(20) to (24).

(26) An immunosuppressant or a fibrosis inhibitor which comprises acompound mentioned in any of the above-mentioned (19) to (24).

(27) The pharmaceutical according to any of the above-mentioned (1) to(8), which comprises ethyl 2-ketoglutarate or benzyl 2-ketoglutarate.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Firstly, the present invention provides a method for the inductionof effector macrophage which is induced and activated corresponding tothe lesion inherent to the tissues after the organic damage and resultsin progressive lesion after the organic damage.

Thus, the present invention provides (a) a method for the induction ofeffector macrophage which is a cause of glomerular lesion of kidney,lesion of islets of Langerhans of pancreas or diabetes mellitus anddiabetic glomerular lesion, characterized in that, lipopolysaccharide isbrought into contact with human PBMC (hereinafter, abbreviated as “LPSinducing method”) and (b) a method for the induction of effectormacrophage which is a cause of progressive tubulointerstitial lesionafter renal damage, lesion of exocrine, acinar or ductal interstitialtissues of pancreas or pancreatitis and tubulointerstitial lesion,characterized in that, human PBMC are brought into contact withmitomycin-treated human PBMC (hereinafter, abbreviated as “allo-MLCinducing method”).

Hereunder, an LPS inducing method will be illustrated in detail.

With regard to a lipopolysaccharide used for the LPS inducing method,that which has been known per se may be used. For example, thelipopolysaccharide derived from Gram-negative bacteria such asSalmonella and Escherichia coli may be used. It may be either theso-called rough type or smooth type.

For the preparation of the lipopolysaccharide, a method which has beenknown per se may be used. An example is a method where it is extractedfrom microbe and, if desired, a treatment for removing the toxicity isconducted. Examples of the method for extracting from microbe are amethod of extracting with hot phenol (Westphal & Jann., MethodsCarbohydr. Chem., 5, 83-99 (1965)) and a method where microbe is treatedwith proteinase K in the presence of sodium lauryl sulfate. In addition,chemically synthesized one may be used or the commercially available onemay be appropriately used.

In the present invention, it is preferred that lipopolysaccharide isused as a solution and, when it is made into a solution using anappropriate solvent, preferably, RPMI 1640 liquid, it is preferred touse a solution of a high concentration of about 60 to 100 μg/ml, morepreferably about 70 to 90 μm/ml or still more preferably about 80 μg/ml.

The human PBMC can be obtained from human peripheral blood by a methodknown per se. An example for a method for separating the mononuclearcells from human peripheral blood is a method by a centrifugalseparation using Ficoll-Paque (registered trademark; manufactured byPharmacia Fine Chemicals). To be more specific, the above-mentionedmethod comprises (a) a step where Ficoll-Paque is placed at the bottomof a test tube, (b) a step where a blood sample as it is or after beingdiluted is carefully transferred onto the Ficoll-Paque using a pipette,(c) a step where the blood preparation prepared by Ficoll-Paque in (b)is centrifuged at about 400˜500 G for about 30˜40 minutes so that ablood component having larger specific gravity than the specific gravityof Ficoll-Paque comes into Ficoll-Paque or passes Ficoll-Paque and (d) astep where the mononuclear cell layer separated on the upper area ofFicoll-Paque is collected.

A specific mode for the LPS inducing method is a method where human PBMCis incubated in RPMI 1640 medium in the presence of lipopolysaccharideand human AB type serum to induce the effector macrophage and thatmethod is advantageously used in the present invention.

At that time, a combination of any two of or all of lipopolysaccharide,human AB type serum and human PBMC may be previously mixed and thenadded to the medium or each of them may be added to the medium solely.There is no limitation for the order of adding to the medium. Morepreferably however, human untreated PBMC are added to RPMI 1640 mediumto which human AB type serum is previously added and thenlipopolysaccharide is added thereto.

RPMI 1640 medium is mentioned in Goding, J. W. (1980) J. Immunol.Methods, 39, 185 and JAMA, 199 (1957), 519. Alternatively, acommercially available product (manufactured by Sigma) may be used aswell.

More preferred mode for carrying out the LPS inducing method is asfollows.

Human untreated PBMC are dissolved in a concentration of about 2×10⁶cells/ml to RPMI 1640 medium to which gentamicin, L-glutamine and humanAB type serum are added to concentrations of about 5 μg/ml, about 2 mMand about 10% by weight, respectively and then lipopolysaccharide isadded thereto so as to make its final concentration about 80 μg/ml toprepare a culture liquid. The said culture liquid is incubated at about37° C. for about six days with about 5% of CO₂.

A method for the induction of allo-MLC will be mentioned in detail asfollows.

The mitomycin-treated human PBMC used in an allo-MLC inducing method canbe prepared, for example, by adding mitomycin to the human PBMC obtainedby the above-mentioned known method to make the final concentration ofmitomycin about 40 μg/ml followed by subjecting to a heating treatmentat about 37° C. for about 30 minutes.

A specific mode for an allo-MLC inducing method is a method wheremitomycin-treated human PBMC and normal human untreated PBMC aresubjected to a mixed culture in RPMI 1640 medium in the presence ofhuman AB type serum to induce the effector macrophage and that method isadvantageously used in the present invention.

At that time, a combination of any two of or all of human AB type serum,mitomycin-treated human PBMC and human PBMC may be previously mixed andthen added to the medium or each of them may be added to the mediumsolely. There is no limitation for the order of adding to the medium. Itis preferred however that normal human untreated PBMC are added to RPMI1640 medium to which human AB type serum is previously added and thenmitomycin-treated human PBMC are added thereto.

A more preferred mode of carrying out the allo-MLC inducing method willbe as follows.

Human untreated PBMC are dissolved in a concentration of about 2×10⁶cells/ml to RPMI 1640 medium to which gentamicin, L-glutamine and humanAB type serum are added to concentrations of about 5 μg/ml, about 2 mMand about 10% by weight, respectively and then mitomycin-treated humanPBMC are added thereto so as to make the final concentration of about2×10⁶ cells/ml to prepare a culture liquid. The said culture liquid isincubated at about 37° C. for about six days with about 5% of CO₂.

(2) The present invention then provides a method for screening acompound which is able to prevent, mitigate or treat the progressivelesion after organic damage by a selective suppression of induction ofeffector macrophage corresponding to lesion inherent to the tissuesafter organic damage.

Thus, the present invention provides a method for screening a compoundwhich is able to prevent, mitigate or treat glomerular lesion of kidney,lesion of islets of Langerhans of pancreas or diabetes mellitus anddiabetic glomerular lesion, characterized in that, a suppressive actionof a compounded to be tested to the induction of effector macrophagecaused by contact of human PBMC with lipopolysaccharide is measured.

Preferred embodiment of the above-mentioned screening method accordingto the present invention will be as follows.

First, human PBMC are brought into contact with lipopolysaccharide inthe presence of the compound to be tested to induce effector macrophage.Then, measurement is carried out to check whether the compounded to betested shows a suppressive action to induction of effector macrophage.

It is preferred that the above-mentioned induction of effectormacrophage is carried out in the same manner as in the above-mentionedLPS inducing method except that, when lipopolysaccharide is brought intocontact with human PBMC, the compound to be tested is further present.

It is preferred that the measurement of the above-mentioned suppressiveaction to the induction of effector macrophage is carried out bymeasuring the numbers of spontaneous plaque-forming cells (hereinafter,referred to as SPFC) which are produced by bringing the induced effectormacrophage into contact with monolayered autologous erythrocytes. Thus,when the numbers of the produced SPFC are less than the case where notest compound to be tested is present, it is judged that induction ofeffector macrophage is suppressed.

More preferred embodiment will be as follows.

Human untreated PBMC are dissolved in a concentration of about 2×10⁶cells/ml to RPMI 1640 medium to which gentamicin, L-glutamine and humanAB type serum are added to concentrations of about 5 μg/ml, about 2 mMand about 10% by weight, respectively and then lipopolysaccharide isadded to the solution so as to make its final concentration of about 80μg/ml.

A compound to be tested is dissolved in an appropriate solvent,preferably dimethyl sulfoxide (DMSO) of about 0.001% by weight and theresulting test solution is added to above-mentioned medium. At thattime, there are prepared various media where concentrations of thecompound to be tested are different. It is preferred that theconcentrations of the compound to be tested are about 1 μM to 0.001 μM.

The mixture is incubated at about 37° C. for about six days in thepresence of about 5% of CO₂ to induce effector macrophage. The effectormacrophage generated in the cultured PBMC induced from the cultureliquid is recovered. In the recovering, known methods may be used and,for example, there is a method where the adhered thing is recoveredusing a rubber-policeman (spatula made of rubber). After that, washingmay be carried out. For the washing, it is preferred to use a Hankssolution to which gentamicin is added so as to make its concentration ofabout 5 μg/ml.

In the meanwhile, monolayered autologous erythrocytes are prepared. Themonolayered autologous erythrocytes may be manufactured by methods knownper se but the following method is preferred.

Autologous erythrocytes are made about 4% by weight concentration by aHanks solution without serum supplementation. It is preferred to use theautologous erythrocytes which are prepared by the above-mentioned knownmethod and preserved at about 4° C. in a phosphate-bufferedphysiological saline (hereinafter, referred to as PBS) with addition of0.1% of AB serum. Poly-L-lysine is added to a Terasaki plate, treated atabout 37° C. for about 20 minutes and washed with PBS, theabove-mentioned autologous erythrocytes are added immediately thereafterand allowed to stand at about 37° C. for about 30 minutes and theerythrocytes which are not adhered are removed to give a Terasaki plateto which monolayered autologous erythrocytes are adhered.

The induced effector macrophage which is recovered hereinabove isdissolved/suspended in the Hanks solution with 5 μg/ml of gentamicin soas to make about 2×10⁶ cells/ml. To the above Terasaki plate to whichthe monolayered autologous erythrocytes are adhered is added 1 to 10 μlof the Hanks solution, the above dissolved/suspended liquid is addedthereto in the same amount and the mixture is allowed to stand at about37° C. for about 2 hours. After completion of the reaction, it ispreferred to fix by formalin.

Numbers of the produced SPFC can be easily measured by a phase-contrastmicroscope.

With regard to an index for the suppression of induction of effectormacrophage, it is preferred to use an IC₅₀ concentration. The IC₅₀ canbe calculated as follows.

When plural experiments are carried out under the same conditions, amean value of the SPFC production numbers measured as above isdetermined. Then, SPFC production numbers per 1×10⁶ of inducedmacrophages are calculated and, from the recovered induced macrophagenumbers, the produced SPFC numbers are determined (this value will becalled S1). Effector macrophage is induced as above without addition ofthe compound to be tested and SPFC production numbers are measured bythe same manner (this value will be called S2). Concentration of thesubstance to be tested when S1 becomes one half of S2 is defined asIC₅₀.

With regard to the compound which is able to prevent, mitigate or curethe progressive lesion after organic damage found by the screeningmethod according to the present invention, the compound where IC₅₀ is 1μM or less is preferred.

The present invention further provides a method for screening a compoundwhich is able to prevent, mitigate or treat progressivetubulointerstitial lesion after renal damage, lesion of exocrine, acinaror ductal interstitial tissues of pancreas or pancreatitis, and lesionof tubulointerstitial tissues associated with pancreatitis,characterized in that, a suppressive action of a compounded to be testedto the induction of effector macrophage caused by contact of human PBMCwith mitomycin-treated human PBMC is measured.

A preferred embodiment of the above-mentioned screening method accordingto the present invention is as follows. Firstly, human PBMC are broughtinto contact with mitomycin-treated human PBMC in the presence of acompound to be tested to induce effector macrophage. After that, ameasurement is carried out to check whether the compound to be testedshows a suppressive action to the induction of effector macrophage.

It is preferred to induce the above effector macrophage in the samemanner as in the case of inducing method for the above allo-MLC exceptthat the compound to be tested is further present in bringing the humanPBMC into contact with mitomycin-treated human PBMC. Concentration ofthe compound to be tested is preferably in about 1 μM to 0.001 μM thesame as in the above screening method and it is preferred to conduct theinduction of effector macrophage by adding the compounded to be testedin various concentrations.

It is preferred that the measurement of the suppressive action of thecompound to be tested to induction of the above-mentioned effectormacrophage is carried out by the same manner as in the above-mentionedscreening method.

The above-mentioned compound to be tested may be anything and may be,for example, peptide, protein, non-peptidic compound, syntheticcompound, fermented product, cell extract, vegetable extract, animaltissue extract and plasma. It may be either a known compound or a novelcompound.

It is also possible to combine known methods in such a manner thatplural compounds to be tested are screened at the same time and, onlywhen a suppressive action to the induction of effector macrophage isdetected, suppressive action for each compound is measured by theabove-mentioned method and the compound having a suppressive action isidentified, etc.

The present invention further provides a screening kit for carrying outthe above-mentioned screening method. There is no particular limitationfor the form of the said screening kit although the forms which havebeen known per se may be used.

For example, a preferred embodiment of a screening kit for screening acompound which is able to prevent, mitigate or treat glomerular lesionof kidney, lesion of islets of Langerhans of pancreas or diabetesmellitus and diabetic glomerular lesion is a kit which comprises (a)human PBMC, (b) lipopolysaccharide, (c) human AB type serum, (d) RPMI1640 medium and (e) a plate to which monolayered autologous erythrocytesare adhered.

Preferred one is such a kit containing Hanks solution to which (a) asolution prepared by adding human PBMC and lipopolysaccharide are addedto an extent of about 2×10⁶/ml and about 80 μg/ml, respectively to RPMI1640 containing about 5 μg/ml of gentamicin, about 2 mM of L-glutamineand about 10% by weight of human AB type serum, (b) a plate to whichmonolayered erythrocytes are adhered and (c) about 5 μg/ml of gentamicinare added.

A preferred embodiment of a screening kit for screening a compound whichis able to prevent, mitigate or treat progressive tubulointerstitiallesion after renal damage, or lesion of exocrine, acinar or ductalinterstitial tissues of pancreas is a kit comprising (a) human PBMC, (b)mitomycin-treated human PBMC, (c) human AB type serum, (d) RPMI 1640medium and (e) a plate to which monolayered autologous erythrocytes areadhered.

Preferred one is the said kit containing a Hanks solution to which (a) asolution prepared by adding human PBMC and mitomycin-treated human PBMCto an extent of about 2×10⁶/ml each to RPMI 1640 containing about 5μg/ml of gentamicin, about 2 mM of L-glutamine and about 10% by weightof human AB type serum, (b) a plate to which monolayered erythrocytesare adhered and (c) about 5 μg/ml of gentamicin are added.

(3) The present invention provides a pharmaceutical for prevention ortherapy of progressive lesion after organic damage by a selectivesuppression of induction of effector macrophage corresponding to thelesion inherent to the tissues after the organic damage and alsoprovides a therapeutic method using the said pharmaceutical.

Thus, the present invention provides a pharmaceutical for preventionand/or therapy of glomerular lesion of kidney, lesion of islets ofLangerhans of pancreas or diabetes mellitus or diabetic glomerularlesion, characterized in that, the pharmaceutical contains a compoundwhich suppresses the induction of effector macrophage caused by contactof human PBMC with lipopolysaccharide.

The present invention also provides a pharmaceutical for preventionand/or therapy of progressive tubulointerstitial lesion after renaldamage, lesion of exocrine, acinar or ductal interstitial tissues ofpancreas and pancreatitis and lesion of tubulointerstitial tissues whichis a complication of pancreatitis, characterized in that, thepharmaceutical contains a compound which suppresses the induction ofeffector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC.

With regard to (a) a compound which suppresses the induction of effectormacrophage caused by contact of human PBMC with lipopolysaccharide and(b) a compound which suppresses the induction of effector macrophagecaused by contact of human PBMC with mitomycin-treated human PBMC, thecompounds which show the suppression to the induction of effectormacrophage by the above-mentioned screening methods may be exemplified.

When a compound which shows activity as a result of the screening isacidic or basic, a salt of such a compound may be used as thepharmaceutical as well. Salt of the said compound is a salt with aphysiologically acceptable acid (such as inorganic acid and organicacid) or base (such as alkaline metal). To be more specific, there maybe used inorganic acid salt such as a salt with hydrochloric acid,phosphoric acid, hydrobromic acid and sulfuric acid; organic acid saltsuch as a salt with acetic acid, formic acid, propionic acid, fumaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methanesulfonic acid andbenzenesulfonic acid; inorganic base salt such as sodium salt, potassiumsalt and ammonium salt; and organic base salt such as dimethylamine saltand cyclohexylamine salt.

The compound which is used for a pharmaceutical or a therapeutic methodaccording to the present invention may be a prodrug or a derivative ofthe above-mentioned compound as well.

With regard to the compound used as a pharmaceutical according to thepresent invention, the following compounds may be specificallyexemplified.

Specific examples of the compound which are able to prevent, mitigate orcure glomerular lesion of the kidney, lesion of islet of Langerhans ofpancreas or diabetes mellitus or diabetic glomerular lesion bysuppressing the induction of effector macrophage caused by contact ofhuman PMBC with lipopolysaccharide are the following compounds (6-1,(6-2) and (3-2).

Specific examples of the compounds which are able to prevent, mitigateor cure progressive tubulointerstitial lesion after renal damage, lesionof exocrine, acinar or ductal interstitial tissues of pancreas andpancreatitis and lesion of tubulointerstitial tissues which is acomplication of pancreatitis by suppressing the induction of effectormacrophage caused by contact of human PMBC with mitomycin-treated PMBCare the following compounds (9-1), (7-3), (7-5) and (4-2).

With regard to the above-mentioned compounds, there are a compoundrepresented by the formula (1)

and a compound represented by the formula (2).

There is also an optical isomer γ-lactone represented by the formula (3)

or by the formula (4)

(in the formulae, R²¹ is an optionally substituted naphthyl and R²² isan optionally substituted open-chain hydrocarbon group having 1 to 6carbons) and a mixture of such optical isomers.

Among them, the compound where R²¹ is naphthyl group and R²² is methylgroup is preferred.

There is also a compound represented by the formula (5) and apharmacologically acceptable salt thereof.

(in the formula,

(a) R¹ and R² may be the same or different and each is hydrogen, anopen-chain aliphatic hydrocarbon group which may be substituted orinterrupted by an intervening group, an optionally substituted cyclicaliphatic hydrocarbon group, an optionally substituted aryl group, anoptionally substituted heterocyclic group or an optionally substitutedcondensed heterocyclic group; X² is O, S or NR³ in which R³ is hydrogen,oxygen, an open-chain aliphatic hydrocarbon group which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and n is aninteger from 1 to 5 or

(b) X² is O, S or NR³; R¹, R² and R³ each is a substituent representedby the formula R¹⁰—Z—R¹¹— (in which R¹⁰ and R¹¹ may be the same ordifferent and each is an optionally substituted open-chain or cyclichydrocarbon group, an optionally substituted aryl group, an optionallysubstituted heterocyclic group or an optionally substituted condensedheterocyclic group; and Z is an intervening group); and n is an integerfrom 1 to 5).

There is further a compound represented by the formula (6) or apharmacologically acceptable salt thereof.

(in the formula, R¹, X² and n have the same meaning as defined in theabove-mentioned item(4); X¹ is halogen, cyano group, an optionallysubstituted mercapto group, an optionally substituted sulfo group, anoptionally substituted sulfonyl group, an optionally substitutedhydroxyl group, an optionally substituted amino group or an optionallysubstituted phosphoryl group).

There is still further a compound represented by the formula (7) or apharmacologically acceptable salt thereof.

(in the formula, R¹ and X² have the same meaning as defined in theabove-mentioned item (4); X¹ has the same meaning as defined in theabove-mentioned item(5); R⁵ and R⁶ may be the same or different and eachis

(a) hydrogen, an open-chain aliphatic hydrocarbon group which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted heterocyclic group or an optionally substituted condensedheterocyclic group,

(b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in the formula,R¹⁰ and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group.), or

(c) R⁵ and R⁶ is an optionally substituted aromatic ring together withthe carbon atom to which they are attached).

In the compound represented by the formula (3) or (4), R²¹ is anoptionally substituted naphthyl group.

Examples of the substituent are halogen (preferably, fluorine, chlorineand bromine), an oxo group, an alkanoyl group (preferably C₁₋₈), analkanoyloxy group (preferably C₁₋₈), an alkanoylamino group (preferablyC₁₋₈), carboxyl group, an alkoxycarbonyl group (preferably C₂₋₈), ahaloalkylcarbonyl group (preferably C₂₋₈), an alkoxy group (preferablyC₁₋₈), a haloalkoxy group (preferably C₁₋₈), amino group, an alkylaminogroup (preferably C₁₋₈), a dialkylamino group (preferably C₂₋₁₆), acyclic amino group, an alkylaminocarbonyl group (preferably C₂₋₈),carbamoyl group, hydroxyl group, nitro group, cyano group, mercaptogroup, an alkylthio group (preferably C₁₋₈), an alkylsulfonyloxy group(preferably C₁₋₈), an alkylsulfonylamino group (preferably C₁₋₈) andphenyl group.

Further, the naphtyl group may be substituted by such group(s) at one ormore position(s).

The naphthyl group represented by a substituent R²¹ may also besubstituted with open-chain or cyclic hydrocarbon group which will bementioned later in detail. Carbon number(s) of the hydrocarbon groupis/are preferably 1 to 8. The said open-chain or cyclic hydrocarbongroup may be substituted, for example, with halogen, hydroxyl group,amino group, nitro group, cyano group, mercapto group, carbamoyl group,alkanoyl group, alkanoyloxy group or alkanoylamino group.

The open-chain hydrocarbon group as a substituent for naphthyl group maybe interrupted by an intervening group such as —O—, —CO—, —COO—, —S—,—SO—, —SO₂—, —NH—, —NR³—, —NH—CO—, —NR³—CO—, —NH—SO₂—, —NR³—SO₂—, —Si—or phosphoryl group.

The substituent R³ is

(a) hydrogen, oxygen, an open-chain hydrocarbon residue which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group or

(b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in which R¹⁰and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group).

The naphthyl group represented by the substituent R²¹ may be substitutedwith a substituent represented by R¹⁰—Z—R¹¹— (in the formula, R¹⁰ andR¹¹ may be the same or different and each is an optionally substitutedopen-chain or cyclic hydrocarbon group, an optionally substituted arylgroup, an optionally substituted heterocyclic group or an optionallysubstituted condensed heterocyclic group; and Z is an interveninggroup).

In the compound represented by the formula (3) or (4), R²² is anoptionally substituted open-chain hydrocarbon group having 1 to 6carbon(s).

The term “open-chain hydrocarbon group having 1 to 6 carbon(s)” meansthat carbon number(s) therein is/are 1 to 6 and it may be eitherstraight or branched and either saturated or unsaturated.

Its examples are methyl group, ethyl group, propyl group, isopropylgroup, n-butyl group, isobutyl group, tert-butyl group, n-pentyl group,isopentyl group, tert-pentyl group and n-hexyl group.

Examples of the substituent in the open-chain hydrocarbon group arehalogen, hydroxyl group, amino group, nitro group, cyano group, mercaptogroup, carbamoyl group, alkanoyl group, alkanoyloxy group andalkanoylamino group.

In the compounds represented by the formulae (5) to (7), thesubstituents R¹ and R² may be the same or different and each is

(a) hydrogen, an open-chain aliphatic hydrocarbon group which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group or

(b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in which R¹⁰and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group).

The term “open-chain aliphatic hydrocarbon group” in the substituents R¹and R² may be straight or branched and may be saturated or unsaturated.

To be more specific, there may be exemplified an alkyl group such asmethyl group, ethyl group, n-propyl group, isopropyl group, n-butylgroup, isobutyl group, tert-butyl group, n-pentyl group, isopentylgroup, tert-pentyl group, n-hexyl group, 1,1-dimethylpropyl group and3-methyl-3-butenyl group; an alkenyl group such as vinyl group, allylgroup, 1-propenyl group, isopropenyl group, 2-butenyl group,1,3-butadienyl group and 2-pentenyl group; and an alkynyl group such asethynyl group, 2-propynyl group, 1-butynyl group and 2-butynyl group.

It is also possible that both double bond and triple bond are present inone substituent such as 2-penten-4-ynyl.

Carbon number(s) is/are preferably 1 to 8. Especially for thesubstituent R², ethynyl group or 2-propynyl group are preferred.

The “cyclic aliphatic hydrocarbon group” in the substituent R¹ and R²may be saturated or unsaturated or may be cross-linked.

To be more specific, there may be exemplified a cycloalkyl group such ascyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexylgroup, cycloheptyl group, adamantyl group and bicyclo[2.2.1]heptylgroup; and a cycloalkenyl group such as 2-cyclopenten-1-yl group and2,4-cyclopentadien-1-yl group.

Carbon numbers of the cyclic aliphatic hydrocarbon group are preferably3 to 12.

The open-chain or cyclic aliphatic hydrocarbon group in the substituentsR¹ and R² may be substituted with the substituent which will bementioned later. With regard to the position of such a substituent,there is no particular limitation so far as it is chemically allowed.

The open-chain hydrocarbon group as a substituent in the substituents R¹and R² may be interrupted by an intervening group such as —O—, —CO—,—COO—, —S—, —SO—, —SO₂—, —NH—, —NR³—, —NH—CO—, —NR³—CO—, —NH—SO₂—,—NR³—SO₂—, —Si— or phosphoryl group. R³ is

(a) hydrogen, oxygen, an open-chain hydrocarbon residue which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group or

(b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in which R¹⁰and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group).

The “aryl group” in the substituents R¹ and R² is an aromatichydrocarbon group which may be partially saturated and there may beexemplified phenyl group, benzyl group, biphenyl group, indenyl groupand naphthyl group or a partially saturated group thereof such as2,3-dihydroindenyl group and 1,2,3,4-tetrahydronaphthyl group.

Preferred carbon numbers of the aryl group are 6 to 20.

Such an aryl group may be substituted with the substituent which will bementioned later and, with regard to the position of the linkage and theposition of the substituent, there is no particular limitation so far asthey are chemically allowed.

With regard to the substituent R¹ and R², benzyl group is preferred and,with regard to the substituent R², it is also preferred that 4-positionis substituted with fluorine.

With regard to the “heterocyclic group” in the substituent R¹ and R²,there may be exemplified five- to six-membered saturated or unsaturatedring containing 1 to 3 hetero atom(s) selected from nitrogen atom,oxygen atom and sulfur atoms in a ring.

Examples of such a heterocyclic group are an aromatic heterocyclic groupsuch as pyrrolyl group, furyl group, thienyl group, imidazolyl group,oxazolyl group, thiazolyl group, pyrazolyl group, isoxazolyl group,isothiazolyl group, oxadiazolyl group, triazolyl group, indolyl group,benzofuryl group, benzothienyl group, benzimidazolyl group, benzoxazolylgroup, benzothiazolyl group, pyridyl group, pyrimidinyl group, pyrazinylgroup, pyridazinyl group, triazolyl group, tetrazolyl group, quinolylgroup and isoquinolyl group; a partially saturated heterocyclic groupsuch as pyranyl group, 1,2-dihydroquinolyl group,1,2,3,4-tetrahydroquinolyl group, 1,2-dihydroisoquinolyl group,1,2,3,4-tetrahydroisoquinolyl group, dihydrofuryl group anddihydrothienyl group; and a saturated heterocyclic group such aspyrrolidinyl group, piperidinyl group, piperazinyl group, morpholinylgroup, tetrahydrofuryl group and tetrahydrothienyl group.

Such a heterocyclic group may be substituted with the substituent whichwill be mentioned later and, with regard to the position of the linkageand the position of the substituent, there is no particular limitationso far as they are chemically allowed.

With regard to the “condensed heterocyclic group” in the substituent R¹and R², there may be exemplified a case where a five- to six-memberedsaturated, partially saturated or unsaturated ring containing 1 to 3hetero atom(s) selected from nitrogen atom, oxygen atom and sulfur atomin the ring is condensed with a benzene ring or other heterocyclic ring.

With regard to the condensed heterocyclic ring, there may be exemplifiedindole, 3H-indole, isoindole, benzofuran, benzothiophene, 1H-indazole,benzimidazole, benzoxazole, benzothiazole, benzisodxazole,benzisothiazole, quinoline, isoquinoline, quinazoline,1,2-dihydroquinoline, 1,2,3,4-tetrahydroquinoline,1,2-dihydroisoquinoline and 1,2,3,4-tetrahydroisoquinoline.

Such a condensed heterocyclic group may be substituted with thesubstituent which will be mentioned later. With regard to the positionof the linkage and the position of the substituent in case substituentis present, there is no particular limitation so far as they arechemically allowed.

The substituents R¹ and R² may be a substituent represented by theformula R¹⁰—Z—R¹¹—.

R¹⁰ and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed hetero group. With regard to thesubstituent, there may be exemplified a substituent which will bementioned later.

With regard to the “substituent” in the open-chain or cyclic aliphatichydrocarbon group, the aryl group, the heterocyclic group or thecondensed heterocyclic group, a substituent which is conventionally usedin the field of pharmaceuticals may be used.

Examples of the substituent are halogen (preferably, fluorine, chlorineand bromine), an oxo group, an alkanoyl group (preferably C₁₋₈), analkanoyloxy group (preferably C₁₋₈), an alkanoylamino group (preferablyC₁₋₈), carboxyl group, an alkoxycarbonyl group (preferably C₂₋₈), ahaloalkylcarbonyl group (preferably C₂₋₈), an alkoxy group (preferablyC₁₋₈), a haloalkoxy group (preferably C₁₋₈), an alkyl group (preferablyC₁₋₂₀), amino group, an alkylamino group (preferably C₁₋₈), adialkylamino group (preferably C₂₋₁₆), a cyclic amino group, analkylaminocarbonyl group (preferably C₂₋₈), carbamoyl group, hydroxylgroup, nitro group, cyano group, mercapto group, an alkylthio group(preferably C₁₋₈), an alkylsulfonyloxy group (preferably C₁₋₈), analkylsulfonylamino group (preferably C₁₋₈) and phenyl group.

Further, the group may be substituted by such group(s) at one or moreposition(s).

The substituent X² is O, S, NH or NR³. Here, the substituent R³ has thesame meaning as defined above.

n is an integer of 1 to 5 and, preferably, it is 1.

The substituent X¹ is (a) halogen, (b) cyano group, (c) an optionallysubstituted mercapto group, an optionally substituted sulfo group or anoptionally substituted sulfonyl group, (d) an optionally substitutedhydroxyl group, (e) an optionally substituted amino group or (f) anoptionally substituted phosphoryl group.

Examples of the substituent are halogen (preferably, fluorine, chlorineand bromine), an oxo group, an alkanoyl group (preferably C₁₋₈), analkanoyloxy group (preferably C₁₋₈), an alkanoylamino group (preferablyC₁₋₈), carboxyl group, an alkoxycarbonyl group (preferably C₂₋₈), ahaloalkylcarbonyl group (preferably C₂₋₈), an alkoxy group (preferablyC₁₋₈), a haloalkoxy group (preferably C₁₋₈), an alkyl group (preferablyC₁₋₂₀), amino group, an alkylamino group (preferably C₁₋₈), adialkylamino group (preferably C₂₋₁₆), a cyclic amino group, analkylaminocarbonyl group (preferably C₂₋₈), carbamoyl group, hydroxylgroup, nitro group, cyano group, mercapto group, an alkylthio group(preferably C₁₋₈), an alkylsulfonyloxy group (preferably C₁₋₈), analkylsulfonylamino group (preferably C₁₋₈) and phenyl group.

Further, the group may be substituted by such group(s) at one or moreposition(s), if chemically acceptable.

With regard to the above-mentioned optionally substituted mercaptogroup, optionally substituted sulfo group or optionally substitutedsulfonyl group, there may be exemplified benzoylthio group, tosyl group,phenylsulfo group and phenylsulfinyl group.

With regard to the above-mentioned optionally substituted hydroxylgroup, there may be exemplified methoxy, ethoxy, propionyloxy, allyloxy,benzoxy and naphthoxy.

With regard to the above-mentioned optionally substituted amino group,there may be exemplified methylamino group, ethylamino group, anilinogroup and anisidino group.

With regard to the above-mentioned optionally substituted phosphorylgroup, a substituent represented by the formula (9)

or by the formula (10)

(in the formulae, substituents R⁷ and R⁸ may be the same or differentand each is an optionally substituted open-chain or cyclic hydrocarbongroup, an optionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed hetero group)is preferred. There may be exemplified methylphosphoryl group,dimethylphosphoryl group and methylethylphosphoryl group. Carbonnumber(s) of the said phosphoryl group is/are preferably 1 to 20.

In the compound (7), the substituents R⁵ and R⁶ each is (a) hydrogen, anopen-chain hydrocarbon residue which may be substituted or interruptedby an intervening group, an optionally substituted cyclic aliphatichydrocarbon group, an optionally substituted aryl group, an optionallysubstituted heterocyclic group or an optionally substituted condensedheterocyclic group or is (b) a substituent represented by the formulaR¹⁰—Z—R¹¹— (in which R¹⁰ and R¹¹ may be the same or different and eachis an optionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and Z is an intervening group).

To be specific, the above is the same description as that for thesubstituents R¹ and R².

The substituents R⁵ and R⁶ may be the same or different.

The substituents R⁵ and R⁶ may also form an aromatic ring together witha carbon to which they are bonded. There are exemplified the cases wherebenzene, naphthalene or indene is formed.

The said aromatic ring may be substituted. In that case, one or moreposition(s) may be substituted. Examples of the substituent are halogen(preferably, fluorine, chlorine and bromine), an oxo group, an alkanoylgroup (preferably C₁₋₈), an alkanoyloxy group (preferably C₁₋₈), analkanoylamino group (preferably C₁₋₈), carboxyl group, an alkoxycarbonylgroup (preferably C₂₋₈), a haloalkylcarbonyl group (preferably C₂₋₈), analkoxy group (preferably C₁₋₈), a haloalkoxy group (preferably C₁₋₈), analkyl group (preferably C₁₋₂₀), amino group, an alkylamino group(preferably C₁₋₈), a dialkylamino group (preferably C₂₋₁₆), a cyclicamino group, an alkylaminocarbonyl group (preferably C₂₋₈), carbamoylgroup, hydroxyl group, nitro group, cyano group, mercapto group, analkylthio group (preferably C₁₋₈), an alkylsulfonyloxy group (preferablyC₁₋₈), an alkylsulfonylamino group (preferably C₁₋₈) and phenyl group.

Further, the said aromatic ring may be substituted with theabove-mentioned optionally substituted open-chain or cyclic hydrocarbongroup. Preferably, carbon number(s) of the hydrocarbon group is/are 1 to8. The said open-chain hydrocarbon group may be interrupted by anintervening group such as —O—, —CO—, —COO—, —S—, —SO—, —SO₂—, —NH—,—NR³—, —NH—CO—, —NR³—CO—, —NH—SO₂—, —NR³—SO₂—, —Si— or phosphoryl group(where R³ has the same meaning as the above-mentioned definition).

Still further, the said aromatic ring may be substituted with asubstituent represented by the formula R¹⁰—Z—R¹¹— (in the formula, R¹⁰and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocycylic group; and Z is anintervening group).

The compounds represented by the formulae (5) to (7) have an asymmetriccarbon atom and, therefore, two optical isomers can exist. Accordingly,the pharmaceutical according to the present invention may contain one ofthe optical isomers only or may contain a racemate.

There is no particular limitation for the pharmacologically acceptablesalts of the compounds represented by the formulae (5) to (7) and, to bemore specific, there may be exemplified inorganic acid salt such as asalt with hydrochloric acid, phosphoric acid, hydrobromic acid andsulfuric acid; organic acid salt such as a salt with acetic acid, formicacid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaricacid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid and benzenesulfonic acid; inorganic base salt suchas sodium salt, potassium salt and ammonium salt; and organic base saltsuch as dimethylamine salt and cyclohexylamine salt.

With regard to the specific examples of the above-mentioned substituentsin the present invention, the following substituents may be exemplified.

With regard to the above “alkanoyl group”, there may be exemplifiedformyl group, acetyl group, propionyl group, butyryl group and pivaloylgroup.

With regard to the above “alkanoyloxy group”, there may be exemplifiedformyloxy group, acetoxy group, propionyloxy group, butyryloxy group andpivaloyloxy group.

With regard to the above “alkanoylamino group”, there may be exemplifiedacetylamino group, propionylamino group, butyrylamino group andpivaloylamino group.

With regard to the above “alkoxycarbonyl group”, there may beexemplified methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonylgroup, isopropoxycarbonyl group, butoxycarbonyl group, isobutoxycarbonylgroup, tert-butoxycarbonyl group and pentyloxycarbonyl group.

With regard to the above “haloalkylcarbonyl group”, there may beexemplified fluoroacetyl group, difluoroacetyl group, trifluoroacetylgroup, chloroacetyl group, dichloroacetyl group, trichloroacetyl group,bromoacetyl group, dibromoacetyl group, tribromoacetyl group,3-chloropropionyl group and 4-chlorobutyryl group.

The above “alkoxy group” means a straight or branched alkoxy group andthere may be exemplified methoxy group, ethoxy group, propoxy group,isopropoxy group, butoxy group, tert-butoxy group, pentyloxy group,tert-pentyloxy group and hexyloxy group.

The above “haloalkoxy group” means a group where halogen atom issubstituted in the above “alkoxy group” and there may be exemplifiedfluoromethoxy group, difluoromethoxy group, trifluoromethoxy group,chloromethoxy group, dichloromethoxy group, trichloromethoxy group,bromomethoxy group, dibromomethoxy group, tribromomethoxy group,iodomethoxy group, diiodomethoxy group, triiodomethoxy group,2-fluoroethoxy group, 2,2-difluoroethoxy group, 2,2,2-trifluoroethoxygroup, 2-chloroethoxy group, 2,2-dichloroethoxy group,2,2,2-trichloroethoxy group, 2-bromoethoxy group, 2,2-dibromoethoxygroup, 2,2,2-tribromoethoxy group, 3-chloropropoxy group and4-chlorobutoxy group.

The above “alkylamino group” means a group where an amino group issubstituted with an alkyl group and there may be exemplified methylaminogroup, propylamino group, isopropylamino group, butylamino group,isobutylamino group, tert-butylamino group, pentylamino group,isopentylamino group, tert-pentylamino group and hexylamine group.

The above “dialkylamino group” means a group where an amino group isdisubstituted with an alkyl group in which the types of the alkyl groupsmay be the same or different and there may be exemplified dimethylaminogroup, ethylmethylamino group, diethylamino group, methylpropylaminogroup, ethylpropylamino group, dipropylamino group, diisopropylaminogroup, dibutylamino group, diisobutylamino group, di-tert-butylaminogroup, dipentylamino group, dilsopentylamino group, di-tert-pentylaminogroup and dihexylamino group.

The above “cyclic amino group” means a group where an amino group is ina cyclic form where four- to eight-membered cyclic amino groups arepreferred and there may be exemplified azetidinyl group, pyrrolidinylgroup and piperidino group as well as those having oxygen atom, sulfuratom or nitrogen atom as a hetero atom such as morpholino group,thiomorpholino group and piperazinyl group. The nitrogen atom of4-position of the piperazinyl group may bear a substituent such as alower alkyl group or an aryl group.

The above “alkylaminocarbonyl group” means a group where the“alkylamino” moiety is the already-mentioned “alkylamino group” andthere may be exemplified methylaminocarbonyl group, ethylaminocarbonylgroup, propylaminocarbonyl group, isopropylaminocarbonyl group,butylaminocarbonyl group, isobutylaminocarbonyl group,tert-butylaminocarbonyl group, pentylaminocarbonyl group,isopentylaminocarbonyl group, tert-pentylaminocarbonyl group andhexylaminocarbonyl group.

The above “alkylthio group” means a straight or branched alkylthio groupand there may be exemplified methylthio group, ethylthio group,propylthio group, isopropylthio group, butylthio group, tert-butylthiogroup, pentylthio group, tert-pentylthio group and hexylthio group.

The above “alkylsulfonyloxy group” means a straight or branchedalylsulfonyloxy group, and there may be exemplified methylsulfonyloxygroup, ethylsulfonyloxy group, propylsulfonyloxy group,isopropylsulfonyloxy group, butylsulfonyloxy group,tert-butylsulfonyloxy group, pentylsulfonyloxy group,tert-pentylsulfonyloxy group and hexylsulfonyloxy group.

The above “alkylsulfonylamino group” means a group where an amino groupis substituted with a straight or branched alkylsulfonyl group and theremay be exemplified methylsulfonylamino group, ethylsulfonylamino group,propylsulfonylamino group, isopropylsulfonylamino group,butylsulfonylamino group, tert-butylsulfonylamino group,pentylsulfonylamino group, tert-pentylsulfonylamino group andhexylsulfonylamino group.

In a substituent represented by the formula R¹⁰—Z—R¹¹— (in the formula,R¹⁰ and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, aryl group,heterocyclic group or condensed heterocyclic group; and Z is anintervening group), examples of the intervening group are —O—, —CO—,—COO—, —S—, —SO—, —SO₂—, —NH—, —NR³—, —NH—CO—, —NR³—CO—, —NH—SO₂—,—NR³—SO₂—, —Si— are phosphoryl group where R³ has the same meaning asthe above-mentioned definition.

With regard to the above-mentioned substituent, specific examplesthereof are the following substituents.

(a) With regard to a substituent where the intervening group is —O—,there may be exemplified methoxymethyl group, ethoxymethyl group,ethoxyethyl group, propoxymethyl group, propoxyethyl group,isopropoxymethyl group, butoxymethyl group, butoxyethyl group,butoxypropyl group, tert-butoxymethyl group, tert-butoxyethyl group,pentyloxymethyl group, pentyloxyethyl group, pentyloxypropyl group,pentyloxybutyl group, tert-pentyloxymethyl group, tert-pentyloxyethylgroup, hexyloxymethyl group, hexyloxyethyl group, hexyloxypropyl group,hexyloxypropyl group, benzyloxymethyl group and phenoxymethyl group.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(b) With regard to a substituent where the intervening group is —CO—,there may be exemplified acetylmethyl group, acetylethyl group,acetylpropyl group, acetylbutyl group, acetylpentyl group, acetylhexylgroup, propionylmethyl group, butyrylmethyl group, isobutyrylmethylgroup, valerylmethyl group, isovalerylmethyl group, hexanoylmethyl groupand phenylacetylmethyl group.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(c) With regard to a substituent where the intervening group is —COO—,there may be exemplified acetoxymethyl group, acetoxyethyl group,acetoxypropyl group, acetoxybutyl group, acetoxypentyl group,acetoxyhexyl group, propionyloxymethyl group,tert-butyloxycarbonylmethyl group, 1-isobutyryloxyethyl group,1-cyclohexyloxycarbonylethyl group, benzyloxycarbonylmethyl group,phenoxycarbonylmethyl group and pivaloyloxymethyl group.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(d) With regard to a substituent where the intervening group is —S—,there may be exemplified methylthiomethyl group, methylthioethyl group,methylthiopropyl group, methylthiobutyl group, methylthioheptyl group,methylthiohexyl group, methylthioisobutyl group, ethylthiomethyl group,propylthiomethyl group, butylthiomethyl group, heptylthiomethyl group,hexylthiomethyl group, benzylthiomethyl group and phenylthiomethylgroup.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(e) With regard to a substituent where the intervening group is —SO₂—,there may be exemplified methylsulfonylmethyl group, methylsulfonylethylgroup, methylsulfonylpropyl group, methylsulfonylbutyl group,methylosulfonylheptyl group, methylsulfonylhexyl group,methylsulfonylisobutyl group, ethylsulfonylmethyl group,propylsulfonylmethyl group, butylsulfonylmethyl group,heptylsulfonylmethyl group, hexylsulfonylmethyl group,benzylsulfonylmethyl group and phenylsulfonylmethyl group.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(f) With regard to a substituent where the intervening group is —SO—,there may be exemplified methylsulfinylmethyl group, methylsulfinylethylgroup, methylsulfinylpropyl group, methylsulfinylbutyl group,methylsulfinylheptyl group, methylsulfinylhexyl group,methylsulfinylisobutyl group, ethylsulfinylmethyl group,propylsulfinylmethyl group, butylsulfinylmethyl group,heptylsulfinylmethyl group, hexylsulfinylmerhyl group,benzylsulfinylmethyl group and phenylsulfinylmethyl group.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(g) The substituent where the intervening group is —NH— is a compoundrepresented by the formula R¹⁰—NH—R¹¹— where R¹¹ is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group andan optionally substituted condensed heterocyclic group as mentionedalready. Examples of R¹⁰—NH— are methylamino group, ethylamino group,propylamino group, isopropylamino group, butylamino group, isobutylaminogroup, tert-butylamino group, pentylamino group, isopentylamino group,tert-pentylamino group, hexylamino group, anilino group and benzylaminogroup.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(h) The substituent where the intervening group is —NR³— is a compoundrepresented by the formula R¹⁰—NR³—R¹¹— where R¹¹ is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group andan optionally substituted condensed heterocyclic group as mentionedalready.

Examples of R¹⁰—NR³— are dimethylamino group, ethylmethylamino group,diethylamino group, methylpropylamino group, ethylpropylamino group,dipropylamino group, diisopropylamino group, dibutylamino group,diisobutylamino group, di-tert-butylamino group, dipentylamino group,diisopentylamino group, di-tert-pentylamino group, dihexylamino group,dibenzylamino group and methylbenzylamino group.

The substituent R³ has the same meaning as the above-mentioneddefinition.

Preferred carbon number(s) of the said substituent is/are 1 to 10.

(i) With regard to the substituent where the intervening group is—NH—CO—, —NR³—CO—, NH—SO₂— or —NR³—SO₂—, there may be exemplified acompound where an intervening group is changed to the above-mentionedone in the above-mentioned compound where the intervening group Z is—NH— or —NR³—.

(j) With regard to the substituent where the intervening group Z is—Si—, there may be exemplified methylsilylmethyl group, methylsilylethylgroup, methylsilylpropyl group, methylsilylbutyl group,methylsilylheptyl group, methylsilylhexyl group, methylsilylisobutylgroup, ethylsilylmethyl group, propylsilylmethyl group, butylsilylmethylgroup, heptylsilylmethyl group, hexylsilylmethyl group,benzylsilylmethyl group and phenylsilylmethyl group.

(k) The substituent where the intervening group Z is a phosphoryl groupis represented by the formulae;

(in the formulae, the substituents R¹⁰, R¹¹ and R¹² may be the same ordifferent and each is an optionally substituted open-chain or cyclichydrocarbon group, an optionally substituted aryl group, an optionallysubstituted heterocyclic group or an optionally substituted condensedhetero cyclic group). There may be exemplified methylphosphoryl group,dimethylphosphoryl group and methylethylphosphoryl group. Carbonnumber(s) of such a substituent is/are preferably 1 to 20.

All of the above-mentioned compounds according to the present inventionmay be manufactured by known methods or methods similar thereto. Methodsfor the manufacture of the compounds of the present invention will beexemplified as follows.

In the case of the compound represented by the formula (5) where R¹ isnot hydrogen, it can be manufactured by cyclization of a compound, forexample, represented by the formula (11)

(in the formula, R¹, R² and X² are the same as those defined already;and W is a leaving group) by means of an intramolecular ring closingreaction.

Here, preferred leaving groups are the leaving groups which are knownper se such as halogen, ester group, mercapto group, etc.

As to the condition for the intramolecular ring closure reaction, knownconditions may be used. Thus, for example, heating is carried out in anorganic solvent, preferably in toluene, at about 30 to 100° C. or,preferably, about 50 to 70° C. for about 5 to 20 hours or, preferably,about 8 to 15 hours.

After that, the solvent is usually removed. Removal of the solvent maybe carried out under reduced pressure or in vacuo depending uponnecessity.

If desired, purification may be carried out by known methods such aschromatography or filtration to give the compound represented by theformula (5).

In the case of the compound represented by the formula (5) where R¹ ishydrogen, a starting material where R¹ is a protective group such asbenzyl group is firstly prepared and then it is hydrolyzed by a knownmethod or by a method similar thereto.

Depending upon the type of the protective group represented by R¹,catalytic reduction may be carried out instead of hydrolysis. Conditionfor the catalytic reduction may follow the known reaction condition andthere is exemplified a method where the starting material is broughtinto contact with hydrogen gas under ordinary or high pressure in thepresence of a catalyst such as palladium-carbon, palladiumhydroxide-carbon, platinum oxide or palladium black in an amount ofabout 3 to 20% by weight or, preferably, about 5 to 15% by weight.

If desired, purification may be carried out by known methods such aschromatography or filtration to give the compound represented by theformula (5).

In the case of the compound (6) where R¹ is not hydrogen and thesubstituent X² is O, the aimed compound is manufactured by adding anucleophilic agent containing X¹ (definition of X¹ is the same asdefined above) such as (COCl)₂ when X¹ is Cl or by adding a cyanide whenX¹ is CN to a compound represented by the formula (12)

(in the formula, R¹ has the same meaning as defined already) in anorganic solvent, preferably ether.

This reaction is believed to proceed in such a mechanism that anintramolecular ketalization firstly takes place and a nucleophilic agentattacks the resulting hydroxyl group.

After that, there is carried out an after-treatment such as removal ofthe solvent. Removal of the solvent may be carried out either underreduced pressure or in vacuo upon necessity.

If desired, purification may be carried out by known methods such aschromatography or filtration to give the compound represented by theformula (6).

In the case of the compound (7) where R¹ is not hydrogen, thesubstituent X² is O and R⁵ and R⁶ form a benzene ring together with thecarbon atoms bonded thereto, a compound where X¹ is OH is synthesizedby, for example, addition of 1,2-isochroman-1,3,4-trione to benzylalcohol and pyridine.

After that, a nucleophilic agent containing X¹ (definition of X¹ is thesame as defined above) to be manufactured such as (COCl)₂ is added whenX¹ is Cl or, when X¹ is CN, a cyanide is added to substitute the OHgroup with the substituent X² to manufacture the aimed compound.

Such a reaction may be carried out according to conventional means.

The compounds represented by the above-mentioned formulae (1) to (7) maybe metabolized in vivo, for example, as follows. Such metabolites may bea pharmaceutical according to the present invention as well.

(in the formulae, Bn is benzyl group and Et is ethyl group)

The following compound (7-2), (7-3), (7-5) or (7-6) may be metabolizedto a compound represented by the formula (7-1). In some cases, thefollowing compound (7-2), (7-3) or (7-5) may also be metabolized to acompound where an ester group is converted to a carboxyl group.

The pharmaceutical in accordance with the present invention may be in adosage form such as tablets which are, if desired, sugar-coated orfilm-coated, capsules, elixirs or microcapsules and may be administeredorally. Further, the pharmaceutical according to the present inventionmay be a parenteral preparation represented by injection such as anaseptic solution or suspension preparation with water or otherpharmaceutically acceptable liquid.

The above-mentioned preparations may be manufactured by a method whichis known per se.

The pharmaceutical according to the present invention may furthercontain other pharmacologically active component which is effective toprogressive lesion after organic damage.

The pharmaceutical according to the present invention may still furthercontain additives which have been used in the related art such asbinders, disintegrating agents, fillers, antiseptic agents, stabilizersand flavors.

With regard to the additive which may be mixed with tablets or capsules,there may be exemplified those which have been used in the related artsuch as binders, disintegrating agents, fillers, antiseptic agents,stabilizers and flavors. To be more specific, there may be used binderssuch as hydroxypropyl cellulose, hydroxypropyl methyl cellulose,Macrogol, gelatin, corn starch, tragacanth and gum arabic;disintegrating agents such as starch and carboxymethyl cellulosecalcium; fillers such as lactose, starch and crystalline cellulose;swelling agents such as corn starch, gelatin and alginic acid;lubricants such as magnesium stearate and talc; sweeteners such assucrose, lactose and saccharine; and flavors such as peppermint, oilderived from Gaultheria ovatifolia ssp. Adenothrix and cherry. When thedosage form is a capsule, it is also possible to add a liquid carriersuch as fat/oil in addition to the above additives.

With regard to an aqueous solution for injection, it is possible to usea physiological saline and other isotonic solution containing glucoseand other excipients such as D-sorbitol, D-mannitol and sodium chloride.In that case, it is further possible to jointly use the appropriatesolubilization aids such as alcohol (e.g., ethanol), polyalcohol (e.g.,propylene glycol and polyethylene glycol) and nonionic surface-activeagent (e.g., Polysorbate 80™ and HCO-50). In the case of an oily liquidfor injection, sesame oil, soybean oil, etc. may be used. Solubilizingaids such as benzyl benzoate and benzyl alcohol may be also usedtogether therewith. It is also possible to compound with buffer such asphosphate buffer and sodium acetate buffer; anesthetizing agent such asbenzalkonium chloride and procaine hydrochloride; stabilizer (such ashuman serum albumin and polyethylene glycol; preserving agent(antiseptic) such as chlorobutanol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, benzyl alcohol and phenol; antioxidant; etc. Thepharmaceutical preparation such as injection prepared as such is usuallyfilled in appropriate ampoules.

Daily dose of the pharmaceutical of the present invention may varydepending upon the type of the effective ingredient, diseases to betreated, route for administration, dosage form, etc. and is notdefinitely decided. Preferably however, it is about 0.1 to 100 mg/kg,more preferably about 1 to 50 mg/kg and it is alsopossible to be about0.5 to 50 mg/kg. Those compounds are of low toxicity and are able to beadministered either orally or parenterally.

The compounds of the present invention represented by theabove-mentioned formulae (1) to (7) have immunosuppressive or fibrosisinhibiting action. To be more specific, the compound according to thepresent invention exhibits an effect of selectively suppressing theeffector macrophage expressed in the damaged tissue caused by organicdamager or immune disease whereby it is able to selectively exhibit animmunosuppressive action selectively to specific tissues. Further, it isable to effectively inhibit the progress or worsening of the disease bythe said effector macrophage whereby it specifically exhibits a fibrosisinhibiting action selectively to damaged tissues.

Accordingly, the pharmaceutical which contains the compound of thepresent invention represented by the above-mentioned formulae (1) to (7)is able to be used as an immunosuppressant or a fibrosis inhibitor. Tobe more specific, the pharmaceutical can be used for prevention of onsetor progress of rejection upon allogeneic or xenogeneic cell, tissue ororgan transplantation, acute or chronic glomerular nephritis,interstitial nephritis or diabetes mellitus; therapy and/or preventionof complications such as diabetic nephropathy, diabetic retinopathy anddiabetic neuropathy; therapy and/or prevention of chronic pancreatitis,arteriosclerosis, arteriosclerotic restenosis, pulmonary fibrosis,dialytic amyloidosis, chronic hepatitis, cerebrospinal degenerativedisease, asthma, rheumatic arthritis, chronic pigmentary dermatitis,psoriasis, autoimmune chronic organic tissue damage, endotoxin shockreaction by bacterial toxin, systemic intravascular coagulation andcancer or metastasis thereof; and prevention and therapy of aids virusinfection. It may also be used as a substitute for steroidal therapeuticagents.

Daily dose of the above-mentioned pharmaceutical according to thepresent invention used as an immunosuppressants or a fibrosis inhibitormay vary depending upon the type of the effective ingredient, thedisease to be treated, route of administration, dosage form, etc. and isnot definitely decided. Preferably however, it is about 0.1 to 100mg/kg, more preferably about 1 to 50 mg/kg and it is also possible to beabout 0.5 to 50 mg/kg in terms of the compound represented by theformulae (1) to (7). Those compounds are of low toxicity and are able tobe administered either orally or parenterally.

As mentioned above, the pharmaceutical according to the presentinvention which is used as an immunosuppressant or a fibrosis inhibitormay contain other pharmacological components showing animmunosuppressive action or a fibrosis inhibiting action as mentionedabove. Further, it may be in various dosage forms as mentioned aboveand, still further, it may contain known additives depending upon theabove-mentioned dosage form.

In the following Examples, Bn means a benzyl group.

EXAMPLE 1 Manufacture of (RS)-(−)-α-methyl-2-naphthalene-methyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate

To a solution prepared by dissolving 164 mg (0.6 mmol, 1 equivalent) of2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylic acid manufacturedby a method mentioned in paragraph [0018], page 5 of Japanese PatentLaid-Open No. 04/338,331 in 2.2 ml of ether at 0° C. were added 240 μl(2.7 mmol, 4 equivalents) of (COCl)₂ gradually and then two drops ofdimethylformamide (DMF) were added thereto. At that time, discharge ofgas was observed.

The reaction solution was allowed to stand for 1 hour with stirring at0° C., ether was removed in vacuo and the product was dried in vacuo.

The product was dissolved in 2.5 ml of ether, then 130 mg (0.75 mmol,1.1 equivalents) of (S)-(−)-α-methyl-2-naphthalenemethanol and 8 mg(0.07 mmol, 0.1 equivalent) of dimethylaminopyrrolidone (hereinafter,abbreviated as DMAP) were gradually added and, at the same time, 140 μl(1 mmol, 1.5 equivalents) of triethylamine were added thereto as well.

After the reaction, the product is dissolved in 20 ml of ether, thesolution was washed with an aqueous solution of NaHCO₃ and the organicphase was dried over MgSO₄.

The solvent was removed in vacuo and, as a result of the firstpurification by silica gel, 175 mg (yield: 74%) of the product wereobtained. By means of purification using silica gel for several timesthereafter, an optical isomer was separated from a mixture of twodiastereomers.

About the compound of the formula (3), i.e.(S)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate:

Thin-layer chromatography: R_(f)=0.56 (hexane/ether, 1:1 (v/v))

T_(f)=97-98° C.

[α]²³ _(D)=−113 (c=3 in CHCl₃)

RMN ¹H (300 MHz; CDCl₃) d ppm: 1.44 (3H, d, J=6.7 Hz); 2.47-2.8 (4H, m);6.03 (1H, q, J=6.7 Hz); 6.82-6.88 (2H, m); 7.03-7.09 (2H, m); 7.34-7.35(1H, m); 7.49-7.51 (2H, m); 7.73 (1H, s); 7.8-7.9 (3H, m).

RMN ¹³C (75 MHz; CDCl₃) dppm: 21.54; 27.4; 33.1; 75.5; 105.3; 116 (d,²J_(CF)=23 Hz); 120.5 (d, ³J_(CF)=7.2 Hz); 123.8; 125.6; 126.6; 127.8;128.2; 128.7; 133.1; 133.3; 137.2; 150.4; 159.3 (d, ¹J_(CF)=244 Hz);166.0; 174.0.

IR (CsI) ν: 3423; 2981; 1797; 1758; 1504; 1290; 1194; 1165; 1082; 1044;914; 857; 822; 751 cm⁻¹.

About the compound of the formula (4), i.e.(R)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate:

Thin-layer chromatography: R_(f)=0.51 (hexane/ether, 1:1 (v/v))

T_(f)=117-118° C.

[α]²³ _(D)=−20 (c=3 in CHCl₃)

RMN ¹H (300 MHz; CDCl₃) d ppm: 1.62 (3H, d, J=6.7 Hz); 2.49-2.87 (4H,m); 6.05 (2H, q, J=6.7 Hz); 6.76-6.81 (2H, m); 6.82-7.1 (2H, m); 7.17(1H, m); 7.5-7.6 (2H, m); 7.65 (1H, s); 7.7-7.8 (3H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 21.5; 27.41; 33.2; 75.6; 116 (d,²J_(CF)=23 Hz); 120.4 (d, ³J_(CF)=8.6 Hz); 123.8; 125.8; 126.6; 127.7;128.1; 128.6; 133.0; 136.9; 150.3; 159.5 (d, ¹J_(CF)=255 Hz); 166.3;174.0.

IR (CsI) ν: 3423; 2981; 1797; 1758; 1504; 1290; 1194; 1165; 1082; 1044;914; 857; 822; 751 cm⁻¹.

EXAMPLE 2 Manufacture of(±)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylic acid

(a) Manufacture of(−)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylic acid

C₁₁H₉FO₅

Molecular weight=240.1

White solid

T_(f)=128° C.

To a solution of 98 mg (0.24 mmol, 1 equivalent) of(S)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate represented bythe formula (3-1) manufactured in Example 1 dissolved in 4 ml of ethylacetate and 8 drops of ethanol was added a Pd/C catalyst in an amount of10 parts by weight to 100 parts by weight of the above solution. Theresulting solution was washed with water several times, stirred at roomtemperature and subjected to a catalytic reduction using hydrogen for 4hours. The product was dissolved in ethyl acetate, the catalyst wasremoved by filtering through Celite (manufactured by Johns ManvilleSales Co.) and the solvent was removed in vacuo. The product waspurified by means of a reverse phase silica gel chromatography (RP 18)(eluent: acetonitrile/water, 1:1 (v/v)). Acetonitrile was removed invacuo and an aqueous phase was freeze-dried. The present compound waswhite powder and 45 mg (yield: 78%) were prepared.(b) (+)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-3-carboxylic acid

C₁₁H₉FO₅

Molecular weight=240.1

White solid

T_(f)=128° C.

(R)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluoro-phenoxy)-5-oxotetrahydrofuran-2-carboxylate represented bythe formula (4-1) manufactured in Example 1 was subjected to a catalyticreduction with hydrogen in the same manner as above whereupon thepresent compound was selectively manufactured. The present compound waswhite powder and 16 mg (yield: 58%) were obtained.

Compound (1): [a]²³ _(D)=−101 (c=0.6 in MeOH)

Compound (2): [a]²³ _(D)=+116 (c=0.3 in MeOH)

RMN ¹H (300 MHz; CDCl₃) d ppm: 2.63-2.84 (4H, m); 7.08-7.14 (4H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 26.7; 32.6; 105.7; 115.4 (d, ²J_(CF)=23Hz); 120.5 (d, ³J_(CF)=8.6 Hz); 150.9; 159.2 (d, ¹J_(CF)=235 Hz); 168.4;175.1

IR (CsI) ν: 3082; 1775; 1507; 1253; 1199; 1041; 978; 830; 708 cm⁻¹

EXAMPLE 3 Manufacture of benzyl2-(4-fluorobenzyl)-5-oxotetrahydrofuran-2-carboxylate

C₁₉H₁₇FO₄

Molecular weight=328.3

White crystals

T_(f)=70° C.

In the presence of Amberyst resin 15 (manufactured by Rohm & Haas Co.),a solution where 192 mg (0.4 mmol, 1 equivalent) of dibenzyl2-(4-fluorobenzyl)-2-hydroxypentane-1,5-dicarboxylate were dissolved in6 ml of anhydrous toluene was heated at 60° C. for 10 hours. This wasreturned to room temperature and filtered through cotton to remove theAmberyst resin 15. The solvent was removed in vacuo and the product wasseparated by chromatography (eluent: hexane/ethyl acetate, 8.5:2.5(v/v)). After purification by recrystallization from ether-hexane twice,the present compound was obtained as thin flaky crystals in an amount of99 mg (yield: 76%).

Thin-layer chromatography: R_(f)=0.24 (hexane/ethyl acetate, 7:3 (v/v))

RMN ¹H (300 MHz; CDCl₃) d ppm: 2.15-2.52 (4H, m); 3.1 (1H, d, J=14.4Hz); 3.3 (1H, d, J=14.4 Hz); 5.14 (1H, d, J=12 Hz); 5.18 (1H, d, J=12Hz); 6.92 (2H, t, J=8.6 Hz); 7.1 (2H, t, J=8.6 Hz); 7.25-7.36 (5H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 27.8; 30.4; 41.6; 67.6; 86.1; 115.2 (d,²J_(CF)=22 Hz); 128.3; 128.6; 129.5; 131.9 (d, ³J_(CF)=6.8 Hz); 134.7;162.3 (d, ¹J_(CF)=244 Hz); 170.8; 175.3.

IR (CsI) ν: 1784; 1736; 1508; 1223; 1189; 1097; 1056; 973; 910; 840;700; 607 cm⁻¹.

SM (IC/NH₃) m/z (intensite relative): 346=100% (MNH₄ ⁺); 347=23%;674=19% (2×M+NH ₄ ⁺).

EXAMPLE 4 Manufacture of2-(4-fluorobenzyl)-5-oxo-tetrahydrofuran-2-carboxylic acid

C₁₂H₁₁FO₄

Molecular weight=238.2

White crystals

T_(f)=93-94° C.

To a solution prepared by dissolving 71 mg (0.2 mmol, 1 equivalent) ofbenzyl 2-(4-fluorobenzyl)-5-oxo-tetrahydrofuran-2-carboxylaterepresented by the formula (5-1), manufactured by the above Example 3,in a mixture of 1.5 ml of ethyl acetate and 3 drops of ethanol was addeda Pd/C catalyst in an amount of 10 parts by weight to 100 parts byweight of the above solution. The reaction solution was washed withwater several times and subjected to a catalytic reduciton with hydrogenfor 6 hours with stirring. Completion of the reaction was confirmed bymeans of a thin-layer chromatography.

The product was filtered through Celite (manufactured by Johns ManvilleSales Co.) and the solvent was evaporated in vacuo. As a result, thepresent compound in white powder was obtained in an amount of 46 mg.

Thin-layer chromatography: R_(f)=0.23 (ethyl acetate/acetic acid, 98:2(v/v))

RMN ¹H (300 MHz; CDCl₃) d ppm: 2-2.55 (4H, m); 3.12 (H, d, J=14 Hz);3.37 (1H, d, J=14 Hz); 7.01 (2H, t, J=8 Hz); 7.25 (2H, t, J=8 Hz); 8.01(1H, s).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 28.8; 31.9; 42.6; 88.0; 115.7 (d,²J_(CF)=21 Hz); 132.2; 133.3 (d, ³J_(CF)=7.2 Hz); 163.5 (d, ¹J_(CF)=242Hz); 174.2; 178.3.

IR (CsI) ν: 3124; 1769; 1511; 1409; 1270; 1244; 1223; 1178; 1037; 961;933; 848; 773; 696; 658 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 256=100% (MNH₄ ⁺); 257=14%;494=14% (2×M+NH₄ ⁺).

EXAMPLE 5 Manufacture of benzyl2-prop-2-ynyl-5-oxo-tetrahydrofuran-2-carboxylate

C₁₅H₁₄O₄

Molecular weight=258.2

Colorless liquid

In the presence of Amberyst resin 15 (manufactured by Rohm & Haas Co.),a solution of 160 mg (0.43 mmol, 1 equivalent) of dibenzyl2-hydroxy-2-(2-prop-2-ynyl)pentane-1,5-dicarboxylate in 5 ml ofanhydrous toluene was stirred overnight at 60° C.

When the reaction finished, the reaction solution was returned toordinary temperature and spherical Amberyst resin was removed byfiltering through cotton. The solvent was removed in vacuo.

The product was purified by a silica type chromatography (eluent:hexane/ethyl acetate, 7:3 (v/v)). The present compound was colorlessliquid and was obtained in an amount of 85 mg (yield: 77%).

Thin-layer chromatography: R_(f)=0.23 (hexane/ethyl acetate, 7:3 (v/v))

RMN ¹H (300 MHz; CDCl₃) d ppm: 2.08 (1H, t, J=2.6 Hz); 2.44-2.7 (4H, m);2.89 (1H, dd, J=17.3 Hz, J=2.6 Hz); 2.95 (1H, dd, J=17.3 Hz, J=2.6 Hz);5.22 (1H, d, J=12 Hz); 5.28 (1H, d, J=12 Hz); 7.38 (5H, ml).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 27.1; 28.1; 29.5; 67.8; 72.2; 77.0; 83.9;128.1; 128.6; 134.6; 169.8; 175.2. IR (CsI) ν: 3286; 1793; 1746; 1456;1419; 1339; 1261; 1170; 1068; 933; 753; 699 cm⁻¹.

SM (IC/NH₃) m/z (intensite relative): 276=100% (MNH₄ ⁺); 277=19%;534=16% (2×M+NH₄ ⁺).

EXAMPLE 6 Manufacture of benzyl2-benzyl-5-oxo-tetrahydrofuran-2-carboxylate

C₁₉H₁₈O₄

Molecular weight=310.3

White crystals

T_(f)=93-94° C.

In the presence of Amberyst resin 15 (manufactured by Rohm & Haas Co.),a solution of 180 mg (0.43 mmol, 1 equivalent) of dibenzyl2-benzyl-2-hydroxypentane-1,5-dicarboxylate in 6 ml of anhydrous toluenewas stirred overnight at 60° C.

When the reaction finished, the reaction solution was returned toordinary temperature and spherical Amberyst resin was removed byfiltering through cotton. The solvent was removed in vacuo.

The product was purified by a silica type chromatography (eluent:hexane/ethyl acetate, 8:2 (v/v)). The present compound was a white solidand was obtained in an amount of 84 mg (yield: 63%).

Thin-layer chromatography: R_(f)=0.27 (hexane/ethyl acetate, 7:3 (v/v))

RMN ¹H (300 MHz; CDCl₃) d ppm: 2.1-2.5 (4H, m); 3.15 (1H, d, J=14.3 Hz);3.38 (1H, d, J=14.3 Hz); 5.2 (2H, s), 7.2-7.38 (10H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 27.9; 30.1; 42.3; 67.7; 86.2; 127.4;128.3; 128.5; 128.6; 128.7; 130.4; 171; 175.5.

IR (CsI) ν: 1780; 1744; 1457; 1432; 1269; 1174; 1082; 1040; 914 857;758; 701; 604 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 328=100% (MNH₄ ⁺); 329=34.

EXAMPLE 7 Manufacture of 2-benzyl-5-oxotetrahydrofuran-2-carboxylic acid

C12H12O4

Molecular weight=220.2

White crystals

T_(f)=110-111° C.

According to the manufacturing method for2-(3-fluorobenzyl)-5-oxotetrahydrofuran-2-carboxylic acid (Example 4),the present compound in white solid was obtained in an amount of 34 mg(yield: 90%) from benzyl 2-benzyl-5-oxotetrahydrofuran-2-carboylaterepresented by the formula (5-4) manufactured in the above Example 6.

Thin-layer chromatography: R_(f)=0.19 (ethyl acetate/hexane/acetic acid,6:4:0.2 (v/v/v)).

RMN ¹H (300 MHz; CDCl₃) d ppm: 2.07-2.7 (6H, m); 3.16 (1H, d, J=14.3Hz); 3.39 (1H, d, J=14.3 Hz); 7.19-7.35 (5H, m); 7.88 (1H, sl).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 27.9; 29.9; 42.2; 86.0; 127.5; 128.5;130.5; 133.5; 175.5; 176.1.

IR (CsI) ν: 3032; 2938; 1782; 1712; 1185; 1082; 1044; 930; 746; 698cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 238=100% (MNH₄ ⁺); 239=15.

EXAMPLE 8 Manufacture of benzyl2-chloro-5-oxotetrahydrofuran-2-carboxylate

C₁₂H₁₁ClO₄

Molecular weight=254.6

Yellow oil

To a solution of 7.5 g (31.7 mmol, 1 equivalent) of benzyl2-oxohemiglutarate dissolved in 105 ml of ether at 0° C. were added 8.3ml (98 mmol, 3 equivalents) of (COCl)₂ and then DMF (400 μl) was addedas well. At that time, discharge of gas was observed.

The mixture was stirred at 0° C. for 1 hour and then stirred at ordinarytemperature for 2 hours. The temperature was set at 0° C. and theproduct was gradually neutralized with an aqueous solution of K₂CO₃. Theproduct was extracted from ether (2×150 ml). The extracted organic phasewas washed with water. After that, the reaction solution was dried overMgSO₄ and the solvent was removed in vacuo. The residue was added to acolumn filled with silica gel and purified by passing an eluent ofhexane/ethyl (7:3 (v/v)) through the column to give 8 g of the presentinvention product as yellowish oil.

Thin-layer chromatography: R_(f)=0.4 (hexane/ethyl acetate, 7:3 (v/v))

RMN ¹H (200 MHz; CDCl₃) d ppm: 2.48-2.89 (4H, m); 5.23 (2H, s);7.25-7.35 (5H, m).

RMN ¹³C (50 MHz; CDCl₃) d ppm: 26.6; 35.9; 68.6; 96.4; 128.1; 128.6;134.1; 164.6; 172.5.

IR (CsI) ν: 1817; 1762; 1271; 1166; 1088 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 255=13% (MH⁺); 272=100% (MNH₄ ⁺);273=15%.

EXAMPLE 9 Manufacture of benzyl2-fluoro-5-oxotetrahydrofuran-2-carboxylate

C₁₂H₁₁FO₄

Molecular weight=238.2

White crystals

T_(f)=57-58° C.

To a solution of 1 g (4.23 mmol, 1 equivalent) of benzyl2-oxohemiglutarate dissolved in 10 ml of methylene chloride was added asolution of 670 μl (5 mmol, 1.2 equivalents) ofdiethylaminotrifluorosulfonic acid (hereinafter abbrivated as DAST) in 4ml of methylene chloride at 0° C. After mixing, the reaction solutionbecomes dark red and was placed in a darkroom of 4° C. for 48 hours. Theproduct was adsorbed on silica gel and separated by a silica typechromatography (eluent: hexane/ethyl acetate, 8.5:1.5 (v/v)). Thepresent compound was white crystalline and obtained in an amount of 725mg (yield: 72%).

Thin-layer chromatography: R_(f)=0.3 (hexane/ethyl acetate, 8:2 (v/v)).

RMN ¹H (300 MHz; CDCl₃) d ppm: 2.48-2.91 (4H, m); 5.3 (1H, d, J=12 Hz);5.39 (1H, d, J=12 Hz); 7.44 (5H, sl).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 26.1; 30.6 (d, ²J_(CF)=26 Hz); 68.8; 11.2(d, ¹J_(CF)=238 Hz); 128.6; 128.9; 129.0; 134.2; 164.3 (d, ²J_(CF)=36Hz); 173.0.

IR (CsI) ν: 1815; 1764; 1339; 1313; 1199; 1177; 1103; 1058; 973; 908;776; 751; 699 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 256=100% (MNH₄ ⁺); 257=12%.

Incidentally, the compound represented by the formula (6-5)

was able to be manufactured according to the method of Example 4 frombenzyl 2-fluoro-5-oxotetrahydrofuran-2-carboxylate manufactured in thisExample.

EXAMPLE 10 Manufacture of benzyl2-cyano-5-oxotetrahydrofuran-2-carboxylate

C₁₂H₁₁FO₄

Molecular weight=238.2

White crystals

T_(f)=57-58° C.

To a solution where 200 mg (0.78 mmol, 1 equivalent) of benzyl2-chloro-5-oxotetrahydrofuran-2-carboxylate manufactured in Example 8was dissolved in 1.2 ml of tetrahydrofuran (THF) was added at −78° C. asolution of 209 mg (0.78 mmol, 1 equivalent) of cyanotetrabutylammoniumdissolved in 1.5 ml of THF. The reaction solution soon became red. Thiswas stirred at −78° C. for 15 minutes. The reaction solution wasreturned to ordinary temperature and gradually mixed with a mixture of 5ml of ether and 5 ml of water. After that, water was removed by ether(2×15 ml) and an organic phase was collected. The organic layer waswashed with water and dried over Na₂SO₄. After removal of the solvent,the residue was added to a column filled with silica gel and purified byadding an eluent hexane/ethyl acetate (7:3 (v/v)). The brown oilobtained at that time was further purified. The present compound was awhite solid and obtained in an amount of 177 mg (yield: 93%)

Thin-layer chromatography: R_(f)=0.3 (hexane/ethyl acetate, 7:3 (v//v))

RMN ¹H (300 MHz; CDCl₃) dppm: 2.64-2.85 (4H, m); 5.35 (2H, s); 7.4 (5H,ml).

RMN ¹³C (75 MHz; CDCl₃) dppm: 25.9; 32; 69.6; 74.6; 114.6; 128.3; 128.7;129.0; 133.4; 163.8; 172.4.

IR (CsI) ν: 3035; 1815; 1764; 1498; 1456; 1417; 1379; 1271; 1158; 1072;1048; 961; 895; 754; 698 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 263=100% (MNH₄ ⁺); 264=12%;280=24%.

EXAMPLE 12 Manufacture of benzyl1-hydroxy-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate

C₁₆H₁₂O₅

Molecular weight=284.2

White solid

T_(f)=77° C.

To a solution of 500 mg (2.84 mmol, 1 equivalent) of1,2-isochroman-1,3,4-trione dissolved in 6.5 ml of THF were added 292 μl(2.84 mmol, 1 equivalent) of benzyl alcohol and 230 μl (2.84 mmol, 1equivalent) of pyridine. The reaction solution which was yellow at firstbecame transparent after 15 minutes. The reaction solution was stirredat ordinary temperature for 2 hours.

After that, the solvent was removed in vacuo. The product was dissolvedagain in a mixture of chloroform and water and, after that, an aqueousphase was extracted with chloroform (3×20 ml). The organic phase waswashed with 5% by volume of hydrochloric acid and with water. Theorganic phase was dried over Na₂SO₄ and the solvent was removed invacuo. The product was purified by chromatography using silica gel(eluent: hexane/ethyl acetate, 6:4 (v/v)). The present compound was awhite solid and obtained in an amount of 605 mg (yield: 75%).

Thin-layer chromatography: R_(f)=0.23 (hexane/ethyl acetate, 6:4 (v/v)

RMN ¹H (300 MHz; CDCl₃) d ppm: 5.24 (2H, s); 7.15-7.33 (1H, d, J=7.5Hz); 7.64-7.76 (2H, m); 7.9 (1H, d, J=6.7 Hz).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 68.9; 122.8; 125.7; 126.8; 127.7; 128.5;128.6; 131.3; 133.9; 134.7; 144.6; 167.4; 167.8.

IR (CsI) ν: 3384; 1746; 1467; 1277; 1237; 1202; 1157; 1105; 1083; 907;750; 694 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 302=100% (MNH₄ ⁺); 303=22%.

EXAMPLE 13 Manufacture of benzyl1-fluoro-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylate

C₆H₁₁O₄

Molecular weight=302.7

White solid

T_(f)=78° C.

To a solution where 300 mg (1.05 mmol, 1 equivalent) of benzyl1-hydroxy-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate represented bythe formula (7-1) manufactured in the above Example 12 were dissolved in2 ml of methylene chloride was added at 0° C. a solution of 167 μl (1.22mmole, 1 equivalent) of DAST dissolved in 1.5 ml of methylene chloride.After addition, the reaction solution was stirred at 4° C. for 20 hours.The product was dissolved in methylene chloride, directly adsorbed onsilica gel and then purified by silica gel chromatography (eluent:hexane/ethyl acetate, 8:2 (v/v)). The present compound was colorless oiland obtained in an amount of 213 mg (yield: 71%).

Thin-layer chromatography: R_(f)=0.3 (hexane/ethyl acetate, 8:2 (v/v))

RMN ¹H (300 MHz; CDCl₃) d ppm: 5.2 (1H, d, J=12 Hz); 5.3 (1H, d, J=12Hz); 7.29-7.38 (5H, m); 7.68-7.81 (3H, m); 7.95-7.98 (1H, dd, J=6.7 Hz,J=1.8 Hz).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 68.7; 107.2 (d, ¹J_(CF)=238 Hz); 123.4;126.1; 132.6; 135.4; 125.4; 128.2; 128.6; 128.8; 142 (d, ²J_(CF)=20 Hz);163.3 (d, ²J_(CF)=38.9 Hz); 165.7.

IR (CsI) ν: 3035; 1811; 1766; 1605; 1498; 1468; 1456; 1380; 1341; 1296;1256; 1200; 1163; 1129; 1107; 1083; 1046; 965; 905; 749; 688; 594 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 320=100% (MNH₄ ⁺); 305=25%.

EXAMPLE 14 Manufacture of benzyl1-chloro-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylate

C₁₆H₁₁O₄Cl

Molecular weight=302.7

White solid

T_(f)=78° C.

Into 0.5 ml of ether were dissolved 50 mg (0.17 mmol, 1 equivalent) ofbenzyl 1-hydrodxy-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylaterepresented by the formula (7-1) manufactured in the above Example 12.The solution was cooled at 0° C. and 80 μl (0.8 mmol, 5 equivalents) of(COCl)₂ and 3 drops of DMF were added thereto. At that time, dischargeof gas and precipitate of white turbidity were observed. The reactionsolution was returned to ordinary temperature and stirred for 3 hours.After the reaction, the product was dissolved in an aqueous solution ofK₂CO₃, and the aqueous phase was extracted with ether (3×10 ml). Theorganic phase was washed with water and the solution was dried overNa₂SO₄. The solvent was removed in vacuo. There is no need to purify thepresent compound whereupon 51 mg of white solid were obtained.

Thin-layer chromatography: R_(f)=0.4 (hexane/ethyl acetate, 8:2 (v/v)).

RMN ¹H (300 MHz; CDCl₃) d ppm: 5.32 (2H, s); 7.35-7.38 (5H, m);7.66-7.95 (4H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 69.2; 91.9; 124; 124.3; 125.8; 131.8;135.5; 128.2; 128.7; 128.8; 134; 146.3; 163.8; 166.

IR (CsI) ν: 3034; 1804; 1764; 1603; 1468; 1456; 1288; 1237; 1188; 1114;1042; 985; 960; 905; 746; 697 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 286=100%; 320=95% (MNH₄ ⁺);321=22%.

EXAMPLE 15 Manufacture of benzyl3-oxo-1,3-dihydroisobenzofuran-1-carboxylate

C₁₆H₁₂O₄

Molecular weight=268.2

White solid

T_(f)=101° C.

To a solution where 133 mg (0.43 mmol, 1 equivalent) of benzyl1-chloro-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate represented by theformula (7-3) manufactured in the above Example 14 were dissolved in 6ml of benzene were gradually added 122 μl (0.46 mmol, 1.05 equivalents)of Bu₃SnH and 4 mg (0.02 mmol, 0.05 equivalent) ofα,α′-azoisobutyronitrile (AIBN). The reaction solution was stirred at70° C. for 5 hours and then stirred overnight at ordinary temperature.After the reaction, the solvent was removed in vacuo. The product wasplaced in ether and washed with a 10% by volume of KF solution. Theorganic phase was dried over Na₂SO₄ and the solvent was removed invacuo. The residue was purified by a silica gel chromatography (eluent:hexane/ether, 7:3 (v/v)). The present compound was a white solid andobtained in an amount of 26 mg (yield: 23%).

Thin-layer chromatography: R_(f)=0.32 (hexane/ether, 6:4 (v/v))

RMN ¹H (300 MHz; CDCl₃) d ppm: 5.17 (1H, d, J=12 Hz); 5.23 (1H, d, J=12Hz); 5.86 (1H, s); 7.3 (5H, m); 7.65-7.51 (3H, m); 7.83 (1H, d, J=7 Hz).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 68.0; 77.2; 122.7; 126.0; 130.2; 125.0;128.4; 128.7; 128.8; 134.5; 143.9; 166.5; 169.3.

IR (CsI) ν: 3493; 3069; 2966; 1780; 1758; 1601; 1468; 1455; 1378; 1320;1281; 1256; 1215; 1197; 1058; 1040; 949; 895; 755; 734; 697 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 286=100% (MNH₄ ⁺); 287=227%,554=17% (2×M+NH₄ ⁺).

EXAMPLE 16 Manufacture of benzyl1-(4-fluorophenoxy)-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate

C₂₂H₁₅O₅

Molecular weight=378.34

White solid

T_(f)=75° C.

A solution where 450 mg (1.5 mmol, 1 equivalent) of benzyl1-hydroxy-3-oxo-1,3-dihydrobenzofuran-1-carboxylate represented by theformula (7-1) manufactured in the above Example 12 and 532 mg (4.75mmol, 3 equivalents) of 4-fluorophenol were dissolved in 2 ml ofmethylene chloride was heated at 65° C. for 5 minutes. After heating, itwas gradually added drop-by-drop to a solution where 355 mg (1.7 mmol, 1equivalent) of N,N-dicyclohexylcarbodiimide (DCC) were dissolved in 0.8ml of methylene chloride. After addition, the reaction solution washeated to reflux for 2.5 hours and the reaction container was returnedto ordinary temperature followed by diluting with 10 ml of methylenechloride. The precipitate was removed by filtration and the solvent wasremoved in vacuo. The product was purified by chromatography usingsilica gel (CH₂Cl₂/hexane, 6:4 (v/v)). The present compound was a whitesolid and obtained in an amount of 227 mg (yield; 40%).

Thin-layer chromatography: R_(f)=0.27 (CH₂Cl₂/hexane, 6:4 (v/v)).

RMN ¹H (300 MHz; CDCl₃) d ppm: 5.1 (1H, d, J=12 Hz); 5.2 (1H, d, J=12Hz); 6.85-7.17 (4H, m); 7.28-7.34 (5H, m); 7.64-7.92 (4H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 68.5; 103.2; 115.9 (d, ²J_(CF)=23 Hz);121.5 (d, ³J_(CF)=8 Hz); 123.9; 125.8; 128.6; 131.9; 126.2; 128.2;128.5; 134; 134.9; 143.6; 149.4; 159.4 (d, ¹J_(CF)=242 Hz); 164.9;166.9.

IR (CsI) ν: 1790; 1747; 1603; 1506; 1465; 1272; 1258; 1205; 1097; 1040;996; 959; 830; 784; 741; 697 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 396=100% (MNH₄ ⁺); 397=33%.

EXAMPLE 17 Manufacture of1-(4-fluorophenoxy)-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylic acid

C₁₅H₉O₅

Molecular weight=288.2

White solid

T_(f)=143-147° C.

To a solution where 40 mg (0.1 mmol, 1 equivalent) of benzyl1-(4-fluorophenoxy)-3-oxo-1,3-dihydroisobenzofuran-1-carboxylaterepresented by the formula (7-5) manufactured in the above Example 16were dissolved in a mixture of 1.2 ml of ethyl acetate and 2 drops ofethanol was added a Pd/C catalyst in an amount of 10 parts by weight to100 parts by weight of the said solution. After the reaction solutionwas degassed several times, it was subjected to a catalytic reductionwith hydrogen with stirring at ordinary temperature for 1 hour. Thecatalyst was removed by filtering through Celite (manufactured by JohnsManville Sales Co.). The solvent was removed in vacuo. The compound wasa white solid and obtained in an amount of 28 mg.

Thin-layer chromatography: R_(f)=0.16 (ethyl acetate/methanol/aceticacid, 95:0.5:0.1 (v/v/v)).

RMN ¹H (300 MHz; CDCl₃) d ppm: 6.97-7.19 (4H, m); 7.70-7.88 (4H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 105; 116.7(d, ²J_(CF)=23 Hz); 123.3 (d,³J_(CF)=8.6 Hz); 125.3; 126.4; 133.1; 136.3; 127.5; 145.5; 151.2; 160.9(d, ¹J_(CF)=239 Hz); 168.0; 168.2.

IR (CsI) ν: 3448; 1797; 1734; 1501; 1466; 1257; 1188; 1099; 1032; 960;851; 783; 727 cm⁻¹.

SM (IC/NH₃) m/z (relative intensity): 306=100% (MNH₄ ⁺); 307=24% 594=23%(2×M+18).

EXAMPLE 18

Manufacture of the compound of the following formula

C₉H₆O₅

Molecular weight=194.1

White solid

T_(f)=130-140° C.

Isochroman-1,3,4-trione (64 mg, 0.36 mmol, 1 equivalent) was dissolvedin 0.5 ml of water followed by stirring overnight at ordinarytemperature. After that, the reaction solution was frozen and thenfreeze-dried. No purification was necessary and the present compound wasobtained white solid in an amount of 69 mg.

RMN ¹H (300 MHz; CDCl₃) d ppm: 7.61-7.91 (4H, m).

RMN ¹³C (75 MHz; CDCl₃) d ppm: 123.1 (sl); 125.1 (sl); 131.8; 134.6(sl); 168.8 (sl).

IR (CsI) ν: 3494; 3067; 1777; 1745; 1467; 1386; 1285; 1232; 1197; 1163;1104; 1079; 1001; 930; 769; 706 cm⁻¹.

n SM (IC/NH₃) m/z (relative intensity): 212=100% (MNH₄ ⁺); 229=85%.

EXAMPLE 19 Manufacture of (RS)-(+)-α-methyl-2-naphthalene-methyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate

The above two compounds were manufactured by the same manner as inExample 1.

The above γ-lactone derivatives may be classified into the followinggroups A-D when the conventional gamma-lactone immunosuppressivecompound (a racemate of ethyl2-(4-fluorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate mentioned inJapanese Patent Laid-Open No. 04/338,331) is taken as a lead compound.

Group A: compounds where the conventional compound which is racemic isseparated into single compounds which are optical isomers

Group B: compounds where oxocyclic oxygen of the lead compound issubstituted with carbon which are more stable to an enzymatic hydrolysisand show an activity similar to the lead compound.

Group C: compounds where 4-fluorophenoxy group of the lead compound issubstituted with an unstable substituent such as halogen, alkoxy group,benzoxy group, nitrile group, etc. Incidentally, such compounds may alsobe grasped as prodrugs of ketoglutaric acid having activity.

Group D: compounds where an aromatic ring is added to the lead compoundfor stability of the compound and, in addition, 4-fluorophenoxy group orhalogen is introduced for affinity to lipid. Such compounds may also begrasped as a compound represented by the formula (7-1) having activityand prodrugs thereof.

(1) Evaluation of effector macrophage suppressive action showing aselective suppressive action to target cell damage in vitro

A suppressive effect of the test compound to effector macrophageinduction was investigated in vitro by an induction and productionsystem of spontaneous plaque-forming cell (SPEC) mentioned in M.Ishibashi, S. Jiang, Y. Kokado, S. Takahara, T. Sonoda:Immunopharmacologic effects of immunosuppressive agents explored by anew effector generation assay. Transplant Proc., 21:1854-1858, 1989.

TEST EXAMPLE 1

(Step 1: Separation of Human PBMC)

Heparin-added peripheral blood (40 ml) was collected from a healthyperson and the same amount of EDTA-added physiological saline was added.PBMC were obtained by a specific gravity centrifugal method usingFicoll-Hypaque (manufactured by Pharmacia Fine Chemicals). Self-plasmawas added thereto and allowed to stand at 37° C. for 10 minutes andplatelets were removed by a low-speed centrifugal separation. RPMI 1640liquid containing 15 μg/ml of gentamicin and 2 mM L-glutamine was usedfor washing the PBMC. Concentration of the PBMC was adjusted to 2×10⁶cells/ml using the same liquid.

(Step 2: Addition of an Agonist and a Substance to be Tested)

Each 200 μl of a solution containing 2×10⁶ PBMC/ml prepared in the step1 were added to each well of a micro test plate, lipopolysaccharide(hereinafter, abbreviated as LPS) was diluted with the RPMI 1640 liquidto make the final concentration 80 μg/ml and the said LPS solution wasadded to each well of the 200 μl-micro test plate. Further, each 50 μlof human serum of type AB were added so as to make the concentration 10%by weight. Two such micro test plates were prepared.

After that, to the above two micro test plates was added a solutionwhere the test compound was diluted with 1% by weight of DMSO so as tomake the concentration of the test compound 1 μM. Similarly wereprepared each two micro test plates where the final concentrations ofthe test compound were 0.1 μM, 0.01 μM and 0.001 μM.

The culture liquid prepared as such was incubated for 6 days at 37° C.in 5% CO₂.

(Step 3: Recovery of the Incubated PBMC)

The effector macrophage induced after the incubation was all recoveredusing a rubber-policeman (spatula made of rubber). A Hanks solutioncontaining 5 μg/ml of gentamicin was used for washing and, using thesaid solution, there was prepared a solution where the concentration ofthe induced effector macrophage was 2×10⁶ per ml.

(Step 4: Preparation of Monolayered Autologous Erythrocyte-adheredPlate)

With regard to autologous erythrocytes, those which were preserved at 4°C. in a phosphate physiological saline (hereinafter, abbreviated as PBS)to which 0.1% by weight of AB serum and autologous erythrocytes obtainedfrom PBMC separation were used were used. The preserved autologouserythrocytes were washed for three times with a Hanks solution to whichno serum was added and made into a solution of 4% by weightconcentration using the said Hanks solution. Poly-L-lysine was added toa Terasaki plate and washed with PBS every 20 minutes at 37° C., theabove-mentioned autologous erythrocyte solution was added theretoimmediately, the mixture was allowed to stand at 37° C. for 30 minutesand the erythrocytes which were not adhered to the plate were removed toprepare a Terasaki plate to which self-erythrocytes in a monolayer wereadhered.

A Hanks solution (4 μl) was added to the said Terasaki plate, 4 μl of asolution containing the induced effect or macrophage obtained in thestep 3 were added thereto and the mixture was allowed to stand at 37° C.for 2 hours. After completion of the reaction, it was fixed by formalin.Numbers of the produced SPFC which were adhered or phagocytized weremeasured.

(Step 5: Calculation of Total Amount of SPFC Produced)

A mean SPFC production number was calculated from the measured values ofSPFC production when the concentration of the test compound was thesame. SPFC production number per 1×10⁶ of the induced effectormacrophage was calculated and, from the recovered induced effectormacrophage numbers, total SPFC production amount (S2) was calculated.

(Step 6: Judgment of Positive Compound Having the Activity)

As a control, no test compound was added and the same operation as abovewas carried out to calculate the total SPFC production amount (S1). Theconcentration of the test compound in which the total SPFC productionamount when test compound was added (S2) was one half of that when notest compound was added (S1) was defined as IC₅₀. The test compound whenits IC₅₀ was 1 μM or less was judged to be positive.

TEST EXAMPLE 2

The same operation as in Test Example 1 was carried out except that, inthe step 2, mitomycin-treated human PBMC (allogenic MLC; hereinafter,abbreviated as allo-MLC) was added to each well of the micro test plateso as to make its concentration 2×10⁶ per ml.

Here, the allo-MLC used was prepared in such a manner that mitomycin wasadded to PBMC which was prepared by the same manner as in the step 1 ofTest Example 1 so as to make the concentration of mitomycin 40 μg/mlfollowed by allowing to stand at 37° C. for 30 minutes.

The result is shown in the following Table. The term “structurecharacteristic” in the Table means the above-mentioned groups A-D. Withregard to a functional classification of the compound, the compoundwhich selectively suppresses the SPFC production under the conditions ofallo-MLC stimulation or, in other words, the compound which selectivelysuppresses the induction of effector macrophage in the presence ofallo-MLC is classified as group I while the compound which selectivelysuppresses the SPFC production under the conditions of LPS stimulationor, in other words, the compound which selectively suppresses theinduction of effector macrophage in the presence of LPS is classified asgroup II. Further, the compound which selectively suppresses the SPFCproduction under the conditions of both allo-MLC stimulation and LPSstimulation or, in other words, the compound which non-selectivelysuppresses the induction of effector macrophage is classified as groupIII. Each of the “compound” in the Table is the compound bearing theabove-mentioned number.

TABLE 1 SPFC Production SPFC Production Inhibiting InhibitingConcentration Concentration Structure by all-MLC by LPS FunctionalCharac- Stimulation Stimulation Classifi- teristic Compound (μM: IC₅₀)(μM: IC₅₀) cation A (1) 0.01 >1.0 Group I (2) >1.0 1 Group II (3-2) >1.01 Group II (4-2) 0.01 >1.0 Group I B (5-2) 0.1 0.01 Group II (5-4) 0.011 Group I C (6-1) >1.0 0.001 Group II (6-2) not done 0.001 Group II(6-3) 0.1 0.001 Group II D (7-1) 0.1 1 Group I (7-2) >1.0 0.1 Group II(7-3) 0.01 >1.0 Group I (7-5) 0.01 >1.0 Group I (7-6) >1.0 0.01 Group II(7-7) >1.0 0.01 Group II Metabolite (9-1) 0.01 >1.0 Group I ConventionalCompound 0.1 0.1 Group III

In the Table, “Conventional Compound” is a racemic ethyl2-(4-fluorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate.

The optical isomer according to the present invention selectivelysuppressed the SPFC production by allo-MLC stimulation and that by LPSstimulation. The correlation in the biological activity depending uponthe structure as such was confirmed in the two types of optical isomersof “(1) and (2)” and “(3-2) and (4-2)”.

With regard to the compounds (group I) where SPFC production by allo-MLCstimulation is selectively suppressed, i.e., induction of effectormacrophage in the presence of allo-MLC is selectively suppressed, thecompounds represented by the formulae (1), (4-2), (5-4), (7-1), (7-3)and (7-5) correspondeded thereto. With regard to the compounds (groupII) where SPFC production by LPS stimulation is selectively suppressed,i.e., induction of effector macrophage in the presence of LPS isselectively suppressed, the compounds represented by the formulae (2),(3-2), (5-2), (6-1), (6-2), (6-3), (7-2), (7-6) and (7-7) correspondedthereto. There were some compounds where the SPFC production suppressiveactivity was from 10-fold to 100-fold as compared with the conventionalcompound.

(2) Evaluation of immunosuppressive action showing a selectivesuppressive effect on target cell damage in animal experiments

TEST EXAMPLE 3

Obstruction release model after complete obstruction of unilateralureter for 14 days

Experimental models were prepared by a method devised and established byIshibashi (Michio Ishibashi, et al.: The Japanese Journal of Nephrology,42:248, 2000) using male SD rats of 8-9 weeks age and about 280 g. Thus,the rat was laparotomized under anesthetization with ether and ureterwas ligated with 7-0 Nylon at the height of the margin of lower pole ofleft kidney to close the abdomen. On the 14th day after obstruction, theobstruction was released and urinary passage was reconstructed using acuff. Thus, after 14 days, the part of ligated obstructed ureter wasresected, a polyethylene tube of 25 gages (manufactured by NipponSherwood) was used as a cuff and inserted into and retained at the lumenfrom the cut end of the lower normal ureter, then the cuff was alsoretained in the expanded upper ureter and each of them was ligated andfixed by 7-0 Nylon to reconstruct the urinary passage. At the same time,the right kidney at the opposite side was excised. After the release ofthe obstruction, body weight was measured, blood was collected on the2nd day, 5th day and 7th day from the release to measure serumcreatinine, then the rat was sacrificed under anesthetization and theleft obstruction-released kidney was excised. With regard to the excisedkidney, there were carried out measurement of weight of the kidney andpathological test for the kidney. When the compound of the presentinvention was not administered to the model, destruction of renalstructure was observed in pathological and morphological investigationsduring the obstructed period and after release of the obstruction with alapse of time causing thickening of glomerular Bowman's capsule wall,hyperplasia of mesangial cells, glomerular sclerosis, involution ordilation of urinary tubule, cellular infiltration to interstitialtissues and fibrosis. Cellular infiltration did not increase theCD5-positive T cells and CD11b/CD18 (ED8)-positive macrophage becamedominant on the tenth day.

An in vivo biological test was carried out for the γ-lactone derivativesaccording to the present invention using the above models. With regardto the test compound, bulk powder of the test compound was dissolved inan aseptic physiological saline together with gum arabic and the finalconcentration of gum arabic was 5% (vol/vol). The concentration of thetest compound 30 mg/ml was suspended in 5% gum arabic-saline solution.The preparation was subcutaneously injected at the dose of 30 mg/kgevery day throughout the experiment. With regard to the test compound,there were used (9-1), (4-2) and (7-3) as the compounds of group Isuppressing the SPFC production by allo-MLC stimulation, i.e., thecompounds which selectively suppress the induction of effectormacrophage in the presence of allo-MLC while there was used (3-2) as thecompound of group II suppressing the SPFC production by LPS stimulation,i.e., the compounds which selectively suppress the induction of effectormacrophage in the presence of LPS. Further, with regard to the compoundof group III which suppresses the SPFC production by both allo-MLCstimulation and LPS stimulation, i.e., the compound whichnon-selectively suppresses the induction of effector macrophage, therewas used a racemate of ethyl2-(4-flyuorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate which is theknown compound.

The result is given in the following Table. In the Table, “functionalclassification” of the compound means the above group I, group II andgroup III. “Compound” means the compound bearing each of theabove-mentioned compound numbers. The fact whether glomerular lesion wassuppressed was judged by the ameriolation of the lesions such asthickness of wall of Bowman's capsule, hyperplasia of mesangial cellsand glomerular sclerosis. The fact whether lesion of tubulointerstitialtissue was suppressed was judged by the ameriolation of the lesions suchas thickness of basement membrane of tubules, cellular infiltration andfibrosis of interstitial tissue. In the table, “control” is the resultwhen the same test as above was carried out using 5% gum arabic onlywhich is a solvent.

TABLE 2 Serum Creatinine Rate of Rate of Cases Weight of level (mg/dl)on Cases where where tubulo- kidney (g) on the 7th Day from Glomerularinterstitial the 7th Day Functional Case Release of Lesion was Lesionwas from Release Classification Compound Numbers Obstruction SuppressedSuppressed of Obstruction Group I   (9-1) n = 3 2.0 ± 0.2  0% (0/4) 100%(4/4) 3.01 ± 0.56 Control n = 3 2.0 ± 0.2  0% (0/3)  33% (1/3) 2.62 ±0.70 (4-2) n = 4 2.7 ± 0.3 50% (2/4) 100% (4/4) 3.94 ± 0.32 Control n =4 2.7 ± 0.9  0% (0/4)  0% (0/4) 4.68 ± 0.66 (7-3) n = 4 2.0 ± 0.6 25%(1/4) 100% (4/4) 2.72 ± 0.47 Control n = 4 2.7 ± 0.9 25% (1/4)  25%(1/4) 2.69 ± 0.40 Group II  (3-2) n = 5 2.0 ± 0.4 100% (5/5)   0% (5/5)4.36 ± 0.95 Control n = 3 2.6 ± 0.9 33% (1/3)  0% (0/3) 5.61 ± 1.08Group III Known n = 4 2.2 ± 0.2 75% (3/4)  75% (3/4) 2.73 ± 0.41 CompdControl n = 4 3.1 ± 0.8  0% (0/4)  0% (0/4) 2.21 ± 0.50

Both (9-1), (4-2) and (7-3) as the compounds of the group I whichsuppress the SPFC production by allo-MLC stimulation, i.e., thecompounds which selectively suppress the induction of effectormacrophage in the presence of allo-MLC and (3-2) as the compound of thegroup I which suppresses the SPFC production by LPS stimulation, i.e.,the compound which selectively suppresses the induction of effectormacrophage in the presence of SPFC showed a selective suppressiveeffect. Thus, the group I dominantly suppressed the lesion oftubulointerstitial tissue. The (3-2) of the group II did not suppressthe lesion of tubulointerstitial tissue but suppressed the glomerularlesion only. The conventional compound belongs to the group III and,although it showed suppression, no selective suppression was observed.

In order to further confirm whether the preventive and therapeuticeffect of the γ-lactone derivatives of the present invention to thetarget cell damage is selective, (7-3) of the group I and (6-1) of thegroup II were used as test compounds and they were subcutaneouslyinjected at the dose of 30 mg/kg every day and compared with the controlgroup. With regard to the control group, only 5% gum arabic which is asolvent was used and the same test as above was carried out.

The result is shown in the following Table wherefrom it is apparentthat, in all cases combined with group I and group II compounds, lesionof glomerulus and of tubulointerstitial tissue were suppressed.

TABLE 3 Rate of Weight Cases where of Kidney Creatinine Rate of Lesionof (g) on 7th level (mg/dl) Cases where Tubulo- Day from Combination on7th Day Glomerular interstitial Release of Case from Release Lesion wasTissue was of compounds Numbers of Obstruction Suppressed SuppressedObstruction Combined n = 3 2.1 ± 0.3 100% (3/3) 100% (3/3) 2.27 ± 0.16use of (7-3) and (6-1) Control n = 5 2.5 ± 0.7  0% (0/5)  20% (1/5) 2.55± 0.45

TEST EXAMPLE 4 Evaluation Using Puromycin Chronic Nephrosis Model in Rat

Male SD rat of 8 weeks age of about 250 g was used and 50 μg/kg ofpuromycin were intravenously administered once according to a method ofDiamond, et al. (J. R. Diamond, I. Pesek, S. Ruggeri, M. J. Karnovsky:Essential fatty acid deficiency during acute puromycin nephrosisameliorates late renal injury. Am. J. Physiol, 257:F798˜F807, 1989).There was induced a chronic puromycin nephrosis rat where albuminuriaincreased from about tenth to twelfth week. After eighth week fromadministration of puromycin, each of compound (9-1) and compound (6-1)was subcutaneously injected at the dose of 30 mg/kg/day everyday untilthe 22nd week. Incidentally, as shown in the above Table 1, (9-1) is acompound which suppresses the SPFC production by allo-MLC stimulationor, in other words, a compound which selectively suppresses theinduction of effector macrophage in the presence of allo-MLC (group I)while (6-1) is a compound which suppresses the SPFC production by LPSstimulation or, in other words, a compound which selectively suppressesthe induction of effector macrophage in the presence of LPS (group II).

As a functional evaluation of kidney, albumin in urine was measuredevery week using a kit for the measurement of albumin. On the 22nd week,the rat was killed and anatomized to observe functional andmorphological changes of kidney.

The result is shown in the following Table. The compound (1-1) (group I)well suppressed the lesion of tubulointerstitial tissues while thecompound (2-1) (group II) well suppressed the glomerular lesion.Functional average daily albuminuria was proportional to themorphological change.

Accordingly, it was found that the compounds of the group I whichsuppressed the induction of effector macrophage by allo-MLC stimulationselectively suppressed the lesion of tubulointerstitial tissues whilethe compounds of the group II which suppressed the induction of effectormacrophage by LPS stimulation selectively suppressed the glomerularlesion.

TABLE 4 Rate of Cases Rate of Cases Albumin in where Glomerular whereLesion of Functional Case Urine Lesion was TubulointerstitialClassification Compound Numbers (mg/day) Suppressed Tissue wasSuppressed Group I  (9-1) n = 6 20, 7, 5, 5, 5, 4 50% (3/6) 83% (5/6)Group II (6-1) n = 6 56, 20, 11, 7, 4, 3 67% (4/6) 17% (1/6)

Correlation between the structural characteristic of immunosuppressiveγ-lactone derivative and biological activities in vitro and in vivo isgiven.

TABLE 5 Activity in vitro Activity in vitro Inhibiting ConcentrationInhibiting Concentration Suppressive Suppressive Effect Structure- forSPEC Production by for SPEC Production by Effect to to Lesion ofCharacteristic allo-MLC Stimulation LPS Stimulation GlomerularTubulointerstitial Experimental of compound Compound (μM: IC₅₀) (μM:IC₅₀) Lesion Tissue Model of Rat A (3-2) >1.0 1 ∘ x UUO/Release (4-2)0.01 >1.0 Δ ∘ UUO/Release C (6-1) >1.0 0.001 ∘ x CPN (6-2) not done0.001 ∘ x UUO/Release D (7-3) 0.01 0.01 x ∘ UUO/Release Metabolite (9)0.01 >1.0 x ∘ UUO/Release Conventional Compound 0.1 0.1 ∘ ∘ UUO/Releaseand CPN ∘: available Δ: somewhat available x: not available UUO/Release:ureter was obstructed for 14 days followed by releasing CPN: chronic PANnephrosis

As such, the γ-lactone derivatives in accordance with the presentinvention are able to selectively suppress the induction of effectormacrophage by allo-MLC stimulation and the induction of effectormacrophage by LPS stimulation. It has been also proved that, as a resultthereof, a suppressive effect depending upon the lesion is able to beachieved.

Accordingly, pharmaceutical preparations containing the γ-lactonederivatives according to the present invention can be applied dependingupon the lesions. When, for example, the progressive lesion after therenal damage comes to glomerulus in the case of renal diseases,administration of the pharmaceutical containing the compound of thegroup II is effective. On the other hand, when the progressive lesionafter the renal damage comes to tubulointerstitial tissue,administration of the pharmaceutical containing the compound of thegroup I is effective. When the progressive lesion after the renal damagecomes to both glomerulus and tubulointerstitial tissue, combination useof the pharmaceutical containing the compounds of the above-mentionedgroups I and II is effective. When the progressive lesion afterpancreatic damage comes to islets of Langerhans in the case of thepancreatic disease, administration of the pharmaceutical containing thecompound of the group II is effective while, when the progressive lesionafter pancreatic damage comes to exocrine, acinar and ductalinterstitial tissue of pancreas, administration of the pharmaceuticalcontaining the compound of the group I is effective. When theprogressive lesion after the renal damage comes to both islets ofLangerhans and exocrine, acinar and ductal interstitial tissue ofpancreas, combination use of the pharmaceutical containing the compoundsof the above-mentioned groups I and II is effective.

As mentioned above, the γ-lactone derivatives in accordance with thepresent invention achieve a selective suppressive action unlike theconventional compounds. As a result of such an action, the γ-lactonederivatives in accordance with the present invention only suppress theactivation of induction of effector macrophage acting in a cytotoxicmanner to damaged organ tissue cells and do not suppress the activationof induction of macrophage participating in the regeneration of tissuesand, therefore, they are able to more effectively prevent or cure theprogressive lesion after the organic damage without lowering thedefensive ability of organism.

It has been also found that the compound (9-1), i.e. ethyl2-ketoglutarate, is one of the active metabolite derived from thecompounds of the group I which selectively suppress the SPFC productionby allo-MLC stimulation. Such an active metabolite shows an activity forsuppressing the lesion of in tubulointerstitial tissue in vivo.Similarly, the compound (9-2), i.e. benzyl 2-ketoglutarate, is one ofthe active metabolite of the group II which selectively suppresses theSPFC production by LPS stimulation and is able to suppress the lesion ofglomerulus in vivo.

TEST EXAMPLE 5 Acute and Chronic Toxicity Tests

Toxicity test of benzyl 2-chloro-5-oxotetrahydrofuran-2-carboxylaterepresented by the formula (6-1) manufactured in Example 8 and ethyl2-ketoglutarate represented by the formula (9-1) was carried out. To bemore specific, the former and the latter were subcutaneously injected atthe doses of 30 mg/kg/day and 90 mg/kg/day, respectively for ten daysand their toxicity was investigated whereupon there was only noted anincrease in weights of the liver and the spleen in a light degree.

When both were administered at the dose of 30 mg/kg/day for consecutive14 days, neither reduction of body weight nor death was noted and therewas no abnormality at the subcutaneously injected area whereupon theywere found to be of low toxicity.

PREPARATION EXAMPLE 1

(Tablets) (1) Benzyl 2-chloro-5-oxotetrahydrofuran-2-  10 g carboxylate(2) Lactose  90 g (3) Corn starch  29 g (4) Magnesium stearate  1 g 130g

The components (1) and (2) and 24 g of the component (3) were mixed withwater to granulate, the resulting granules were mixed with 5 g of thecomponent (3) and the component (4), and the mixture was compressedusing a compressive tabletting machine to manufacture 1,000 tablets of 7mm diameter containing 10 mg of the component (1) per tablet.

PREPARATION EXAMPLE 2

(Capsules) (1) Benzyl 2-chloro-5-oxotetrahydrofuran-2-  50 mgcarboxylate (2) Lactose  14 mg (3) Corn starch  29 mg (4) Hydroxypropylcellulose  6 mg (5) Magnesium stearate  1 mg 100 mg per capsule

The above-mentioned components (1), (2), (3) and (4) were mixed andgranulated according to a conventional method. The component (5) wasadded thereto and placed into a gelatin capsule by a conventional methodto give a capsule preparation.

INDUSTRIAL APPLICABILITY

The present invention is able to provide a method for the induction ofeffector macrophage which is induced and activated corresponding to thelesion inherent to the tissues after organic lesion and results in aprogressive lesion after the organic damage.

By utilizing the said method, the present invention is able to provide amethod for screening compounds which are able to prevent, mitigate orcure the progressive lesion after organic damage such as glomerularlesion and tubulointerstitial lesion in the case of kidney and, in thecase of pancreas, exocrine interstitial lesion or pancreatitis andLangerhans islet lesion or diabetes. The present invention also providesa method for screening compounds which are able to prevent, mitigate orcure diabetes and diabetic retinitis or pancreatitis andtubulointerstitial lesion complicated with pancreatitis at the sametime.

It is also possible to provide a pharmaceutical for the prevention orthe therapy of the above-mentioned progressive lesion after the organicdamage or a therapeutic method therefor by utilizing the said screeningmethod. The said pharmaceutical or the said therapeutic method haslittle side effect such as an unnecessary lowering of defense oforganism or induction of new tissue damage and, in addition, it achievesrepair and regeneration of the tissues without unnecessary lowering ofthe defense of organism whereby it is now possible to conduct thetherapy for a long period on a continuous basis using the saidpharmaceutical or therapeutic method.

The novel γ-lactone derivatives according to the present invention arecompounds which are able to selectively suppress the induction ofeffector macrophage corresponding to the lesion inherent to the tissuesafter the organic damage and are able to be used as the above-mentionedpharmaceutical.

The novel γ-lactone derivatives according to the present invention havea selective induction-suppressive action for the above effectormacrophage and, as a result, they show a selective immunosuppressiveaction or a fibrosis inhibiting action to specific tissues. Accordingly,the novel γ-lactone derivatives according to the present invention areable to be used not only as the above-mentioned pharmaceutical but alsoas an immunosuppressant or a fibrosis inhibitor. To be more specific,the pharmaceutical containing the novel γ-lactone derivative of thepresent invention is able to selectively suppress the progress orworsening of the diseases to the target organ or the like and, since itsimmunosuppressive action or fibrosis inhibiting action is strong, it iseffective for therapy and/or prevention of rejection reaction upon ofxenogenic or allogeneic cell, tissue or organ transplantation andendotoxin shock reaction by bacterial toxin, systemic intravascularcoagulation, various inflammatory diseases, chronic inflammatorydiseases and cancer. To be more specific, the pharmaceutical containingthe novel γ-lactone derivative according to the present invention isable to be used for prevention of onset or progress of rejectionreaction upon transplantation of allogeneic or xenogenic organs, cellsor tissues, or acute or chronic glomerular nephritis and onset orprogress of interstitial retinitis or diabetes; therapy and/orprevention of complications such as diabetic nephropathy, diabeticretinopathy and diabetic neuropathy; therapy and/or prevention ofchronic pancreatitis, arteriosclerosis, arteriosclerotic restenosis,pulmonary fibrosis, dialytic amyloidosis, chronic hepatitis,cerebrospinal degeneration, asthma, rheumatic arthritis, chronicpigmentary skin diseases, psoriasis, autoimmune chronic organic tissuedamage, endotoxin shock reaction by cytotoxin, systemic intravascularcoagulation or cancer or its metastasis; and prevention and therapy ofinfection of AIDS virus. It is also able to be used as a substitute forsteroidal therapeutic agent.

1. A pharmaceutical composition, which comprises an active ingredientthat selectively suppresses the induction of effector macrophagesselected from the group consisting of: (i) an optical isomer γ-lactonerepresented by the formula (3):

 or an optical isomer γ-lactone represented by the formula (4):

 wherein, R²¹ is an optionally substituted naphthyl group, R²² is anoptionally substituted straight or branched hydrocarbon residue having 1to 6 carbon atoms, or a mixture of said optical isomers represented bythe formulae (3) and (4); (ii) one of said optical isomers representedby the formula (3) or (4), or a mixture of said optical isomersrepresented by the formulae (3) and (4), wherein R²¹ is naphthyl and R²²is methyl; (iii) a compound represented by the formula (5):

 wherein, (a) R¹ and R² may be the same or different and each ishydrogen, an open-chain aliphatic hydrocarbon group which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; X² is O, S orNR³ in which R³ is hydrogen, oxygen, an open-chain aliphatic hydrocarbongroup which may be substituted or interrupted by an intervening group,an optionally substituted cyclic aliphatic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and n is an integer from 1 to 5; or (b) X² is O, S or NR³, R¹, R²and R³ each is a substituent represented by the formula R¹⁰—Z—R¹¹—,wherein R¹⁰ and R¹¹ may be the same or different and each is anoptionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup, and Z is an intervening group; and n is an integer from 1 to 5,or a pharmacologically acceptable salt thereof; (iv) a compoundrepresented by the formula (6):

 wherein, R¹, X² and n have the same meanings as defined above, X¹ ishalogen, cyano group, an optionally substituted mercapto group, anoptionally substituted sulfo group, an optionally substituted sulfonylgroup, an optionally substituted hydroxyl group, an optionallysubstituted amino group or an optionally substituted phosphoryl group,or a pharmacologically acceptable salt thereof; and (v) a compoundrepresented by the formula (7):

 wherein, R¹, X¹ and X² have the same meanings as defined above, R⁵ andR⁶ may be the same or different and each is (a) hydrogen, a straight orbranched aliphatic hydrocarbon group which may be substituted orinterrupted by an intervening group, an optionally substituted cyclicaliphatic hydrocarbon group, an optionally substituted heterocyclicgroup, or an optionally substituted condensed heterocyclic group; (b) asubstituent represented by the formula R¹⁰—Z—R¹¹, wherein R¹⁰ and R¹¹may be the same or different and each is an optionally substitutedopen-chain or cyclic hydrocarbon group, an optionally substituted arylgroup, an optionally substituted heterocyclic group or an optionallysubstituted condensed heterocyclic group; and Z is an intervening group;or (c) R⁵ and R⁶ together with the carbon atom to which they areattached form an optionally substituted aromatic ring; or apharmacologically acceptable salt thereof.
 2. The pharmaceuticalcomposition according to claim 1, wherein the active ingredient isselected from the group consisting of:

wherein Bn is benzyl.
 3. The pharmaceutical composition according toclaim 1 or 2, wherein the active ingredient is represented by theformula (4-2):


4. The pharmaceutical composition according to claim 1 or 2, wherein theactive ingredient is represented by the formula (6-2):

wherein Bn is benzyl.
 5. The pharmaceutical composition according toclaim 1 or 2, wherein the active ingredient is represented by theformula (7-3):

wherein Bn is benzyl.
 6. The pharmaceutical composition according toclaim 1, wherein the active ingredient is represented by the formula(7), wherein R⁵ and R⁶ together with the carbon atom to which they areattached form an optionally substituted aromatic ring, X² is O, X¹ isoptionally substituted hydroxyl and R¹ is hydrogen.